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u I ~ T I N MARIETTA ENERGY SYSTEMS LIBRARIES
3 4 4 5 6 0 2 7 7 3 b 3 3
ORNL-6355
Expedient Antibiotics Production
Final Report
interagency Agreement DOE 1457-1 457-A 1 and FEMA EMW-84-E- 1737, Work Unit 241 1 K
APPROVED FOR PUBLIC RELEASE; DISTRIBUTION UNLIMITED
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This report was prepared as an account of work sponsored by an agency of the UnitedStatesGovernment. NeithertheUnitedStatesGovernment norany agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United StatesGovernment or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of theUnitedStatesGovernment or any agency thereof.
la . REPORT SECURITY CLASSIFICATION Unclassified
2a. SECURITY CLASSIFICATION AUTHORITY
2b. DECLASSIFICATION I DOWNGRADING SCHEDULE
4 PERFORMING ORGANIZATION REPORT NUMBER(S) N /A
ORNL - 6345
1 b RESTRICTIVE MARKINGS
3 None
DISTRIBUTION 1 AVAILABILITY OF REPORT
Unlimited
S MONITORING ORGANIZATION REPORT NUMBER(S)
N /A
7a. NAME OF MONITORING ORGANIZATION
MARTIN MARIETTA ENERGY SYSTEMS LIBRARIES N / A
6a NAME OF PERFORMING ORGANIZATION
Oak Ridge National Laboratory -., - -
7b ADDRESS (City, Stan, and Zli u1 I llllllllulllul I I ullllllll IllllllUIlll 6b OFFICE SYMBOL
( I f dpplicable)
3 4 4 5 6 0277363 3 N /A
8a. NAME OF FUNDING / SPONSORING ORGANIZATION Federal Emergency
Management Agency
9 PROCUREMENT INSTRUMENT ~OENTIFICATION NUMBER Interagency Ag. EMW-84-E-1737 (FEMA)
1457-1457-A1 (DOE) 10 SOURCE OF FUNDING NUMBERS PROGRAM TASK WORK UNIT
No. ACCESSION NO
8b OFFICE SYMBOL (If dppkdbk)
2411K I 9 I
I I Cephalosporins, Antibiotics, Antibiotic Manufacturing, ' Antimicrobials, Emergency Production of Antibiotics (cont'd
19 ABSTRACT (Continue on reverse if necessary and Identify by block number)
I 19
~UNCLASSIF~ED~UNLIMITED 0 SAME AS RPT 0 OTIC USERS 22a NAME OF RESPONSIBLE INDIVIDUAL
I EXPEDIENT ANTIBIOTICS PRODUCTION I
Unclassified 22b TELEPHONE (Induck Area Code) 22c. OFFICE SYMBOL
1 2 PERSONAL AUTHOR(S) Paul R. Bienkowski, Charles H. Byers, and Douglas D. Lee
Final FROM 1985 TO 1988 April 1988 13a. TYPE OF REPORT 13b TIME COVERED
16 SUPPLEMENTARY NOTATION
14 DATE OF REPORT (Year, Month, Day)
17 COSATI CODES 18 SUBJECT TERMS (Continue on reverse i f necessary and rdentl+ by block number) FIELD I GROUP 1 SUB-GROUP Sulfa, Penicillins, Tetracyclines, Bacitracin,
I I 1
SECURITY ClASSlFlCATlON OF THIS PAGE 00 FORM 1473,84 MAR 83 APR edition may be used until exhausted. All other editions are obsolete.
Antibiotic Manufacturers, Antibiotic Purification and Separation, Alternate Antibiotic Production Sites, Fermentation Equipment, Disease Control, Pharmaceutical Industry
1 9 . ABSTRACT (continued)
I particular antibiotic product. The fermentation section requires specialized equipment
I for operation in a sterile environment which is not usually available in other industries. The emergency production of antibiotics under austere conditions will be feasible only if a substantial reduction in the complexity and degree of separation and purity normally required can be realized. Detailed instructions were developed to assist State and Federal officials who would be directing the resumption of antibiotic production after a nuclear attack. Initially, all plant managers should be contacted (using the lists provided in this report) to determine if their facility has been destroyed or damaged. If a plant has been damaged, the manager should determine the feasibility of making repairs, the potential capacity of the repaired plant, and the type of antibiotic that can be produced. If enough production cannot be realized from undamaged and slightly damaged plants, the plants from the list of alternate emergency production sites must be contacted, determination of the plant status made, and the managers informed of impending conversion to antibiotic production. If alternate sites must be used, a team of skilled personnel must be assembled to convert the plant to antibiotic production in the austere environment.
SECURITY CLASSIfICATION OF THIS PACE
ORNL-6355 Dist. Category UC-41
Chemical Technology Division
EXPEDIENT ANTIBIOTICS PRODUCTION
P. R. Bienkowski C. H. Byers D. D. Lee
Date Published - ?lay 1988
Approved for public release: Distribution unlimited
Prepared for the FEDERAL EMERGENCY MANAGEMENT AGENCY
Washington, DC 20472 Interagency Agreement; FEMA EMW-84-E-1737,
DOE 1457-1457-A1 formerly 40-1457-84 Work Unit No. 2411K
"This report has been reviewed in the Federal Emergency Management Agency and approved f o r publication. does not signify that the contents necessarily reflect the views and policies of the Federal Emergency Management Agency. 'I
Approval
Prepared by the OAK RIDGE NATIONAL LABORATORY Oak Ridge, Tennessee 37831
operated by MARTIN MARIETTA ENERGY SYSTEMS, Inc.
f o r the U.S. DEPARTMENT OF ENERGY
under Contract No. DE-AC05-840R21400
iii
CONTENTS
V LISTOFTABLES . . . . . . . . . . . . . . . . . . . . . . . . LISTOFFIGURES vii . . . . . . . . . . . . . . . . . . . . . . . . EXECUTIVE SUMMARY xi . . . . . . . . . . . . . . . . . . . . . . . .
Objective xi Background . . . . . . . . . . . . . . . . . . . . . . . . . x i Procedure xi Conclusions . . . . . . . . . . . . . . . . . . . . . . . xii
. . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . .
ABSTRACT xvii . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . 1 INTRODUCTION
. . . . . . . . . . . . . . . . . 3 2 DEFINITION OF AN ANTIBIOTIC
3 . ANTIBIOTICS SELECTED FOR EXPEDIENT PRODUCTION . . . . . . . 5
9 3.2 Cephalosporins . . . . . . . . . . . . . . . . . . . 13 3 . 3 Tetracyclines . . . . . . . . . . . . . . . . . . . . 14 3.4 Bacitracin . . . . . . . . . . . . . . . . . . . . . 14 3 . 5 Sulfonamides . . . . . . . . . . . . . . . . . . . . 16
3 . 1 Penicillins . . . . . . . . . . . . . . . . . . . . .
4 . ANTIBIOTIC MANUFACTURERS . . . . . . . . . . . . . . . . . 1 9
5 . ANTIBIOTIC PRODUCTION TECHNOLOGIES . GENERAL DESCRIPTION . 23
5.1 Raw materials and utilities . . . . . . . . . . . . . 25 5.2 Fermentation . . . . . . . . . . . . . . . . . . . . 27 5.3 Separation and purification . . . . . . . . . . . . . 28 5 . 4 Analytic Procedures/ Quality Control . . . . . . . . 30 5 . 5 Advanced Technologies . . . . . . . . . . . . . . . . 30
6 . EXPEDIENT PRODUCTION OF ANTIBIOTICS . . . . . . . . . . . . 33
6.1 Alternate production sites . . . . . . . . . . . . . 33
6 . 3 Single Cell Proteins . . . . . . . . . . . . . . . . 44 6 . 2 Conversion to antibiotic production . . . . . . . . . 33
7 . ALTERNATE SOURCES OF ANTIBIOTICS . . . . . . . . . . . . . 47
8 . REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . 49
8.1 Cited References . . . . . . . . . . . . . . . . . . 49 8.2 General References . . . . . . . . . . . . . . . . . 52 8.3 Penicillins . . . . . . . . . . . . . . . . . . . . . 5 5 8 . 4 Cephalosporin . . . . . . . . . . . . . . . . . . . . 57
iv
8.5 Tetracyclines . . . . . . . . . . . . . . . . . . . . 58
8.8 Separation and Purification . . . . . . . . . . . . . 60
8.11 Analytical Methods . . . . . . . . . . . . . . . . . 64
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.6 Bacitracin 59 8 . 7 Sulfonamides 60
8.9 Preservation 63 8.10 Sterilization 63
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
APPENDIX A Production of Penicillin Antibiotics . . . . . . . 65
APPENDIX B Production of Cephalosporin Antibiotics . . . . . . . 87
APPENDIX C Production of Tetracycline Antibiotics . . . . . . 103
APPENDIX D Sulfonamides . . . . . . . . . . . . . . . . . . . 111
APPENDIX E Bacitracin Production . . . . . . . . . . . . . . . 117
APPENDIX F Organic Product Fermentations . . . . . . . . . . . 125
APPENDIX G Enzyme Production Technology . . . . . . . . . . . 133
APPENDIX H Single Cell Protein Production . . . . . . . . . . 139
APPENDIX I Inoculum Preservation . . . . . . . . . . . . . . . 145
APPENDIX J Sterilization . . . . . . . . . . . . . . . . . . . 151
APPENDIX K Equipment . . . . . . . . . . . . . . . . . . . . . 161
APPENDIX L Separation and Purification . . . . . . . . . . . . 171
High Performance Liquid Chromatography . . . . . . . . . . Supercritical Fluid Extraction . . . . . . . . . . . . . . 180 1 7 3
V
LIST OF TABLES Number Page
1.
2 .
3 .
4.
5 .
6 .
7 .
8 .
9 .
U.S. antibiotic production . . . . . . . . . . . . . . . .
Antibiotics produced in the U.S. (by classification) . . . Dosage forms and dosage recommendations for expedient antibiotics . . . . . . . . . . . . . . . . . . Risk assessment for antibiotic producers in the U . S . assuming three different attack scenarios . . . . . . . .
Growth media for penicillin - shake flask and factory . . Risk assessment for enzyme producers in the U.S. assuming three different attack scenarios . . . . . . . .
Risk assessment for fermentation ethanol producers in the U.S. assuming three different attack scenarios . .
Other candidates for conversion to antibiotic production .
Alternate antibiotic production sites outside of the continental U.S. . . . . . . . . . . . . . . . , . . .
A-1. Raw materials needed for penicillin production . . . . . .
A-2. Alternate materials for defined medium for penicillin production . . . . . . . . . . . . . . . . . .
A-3. Specific uptake rates for nutrients during a penicillin fermentation. . . . . . . . . . . . . . . . . . . . . . .
A - 4 . Carbon distribution into penicillin, mycelium, and maintenance energy . . . . . . . . . . . . . . . . . . .
A - 5 . Maximum and real yields of penicillin from glucose . . . .
A-6. Traditional penicillin purification . . . . . . . . . . .
B - 1 . Cephalosporin production media. . . . . . . . . . . . . . B-2. Carbon utilization for cephalosporin production . . . . .
B-3. Clinical characteristics of common cephalosporin antibiotics . . . . . . . . . . . . . . . . . . . . . . .
B-4. Characteristics of cephalosporins of clinical interest . .
B - 5 . Microorganisms which produce various cephalosporins . . .
6
11
12
20
26
34
36
40
48
68
68
7 1
73
73
78
90
9 1
94
95
97
vi
Number Page
E.1 . Solubility of bacitracins in various solvents . . . . . . 122
E.2 . Precipitating agents for bacitracin . . . . . . . . . . . 1 2 2
K.1 . Suppliers of oxygen probes used in fermenters . . . . . . 170
L.1 . Chromatographic parameters for tetracyclines . . . . . . . 177
L.2 . HPLC assays of antibiotics . . . . . . . . . . . . . . . . 178
L.3 . SCF related physical properties of selected antibiotics . 183
vii
LIST OF FIGURES Number
1.
2.
3 .
4 .
5.
6 .
7.
8 .
9.
10.
A-1.
A-2.
A - 3 .
A-4.
A - 5 .
A - 6 .
A - 7 .
A - 8 .
Structures of Cephalosporin C and Penicillin G produced in antibiotic fermentations . . . . . . . . . . . . . . . . .
Structure of Tetracycline showing substitutions to get related antibiotics . . . . . . . . . . . . . . , . . . .
Chemical Modification to penicillin G to produce semisynthetic penicillins . . . . . . . . . . . . . . . .
Structure of Bacitracin A . For convenience, most of the H atoms in the formula have been omitted . . . . .
Structure of sulfonamides . . . . . . , . . . . . . . . .
Antibiotic producer locations superimposed on U.S. map with two types of nuclear attacks . . . . . . . .
Block diagram for a general antibiotic production process . . . . . . . . . . . . . . . . . . . . . . . . .
Procedure and flowsheet for beginning a penicillin fermentation. . . . . . . . . . . . . . . . . . . . . . .
Enzyme producer locations superimposed on U.S. with two types of nuclear attack . . . . . . . . . . . . .
Fermentation ethanol producer locations superimposed on U . S . map with two types of nuclear attack . . . . . . .
General production scheme for penicillin production . . .
Typical concentration profile of fed-batch penicillin fermentation. . . . . . . . . . . . . . . , . . . . . . . Penicillin purification process of Gist-Brocades . . . . .
Distribution coefficients vs. pH o f penicillin G between water and solvents . . . . . . . . . . . . , . . .
Partition of penicillins as a function of the pH of the extraction solvent (isoamyl acetate). . . . . . . . . . .
Traditional three-stage extraction of penicillin . . . . .
Chemical synthesis of 6-aminopenicilanic acid (6-APA) . .
Chemical structures of some semisvnthetic penicillins . .
map
PaRe
7
8
10
1 5
1 7
22
24
29
38
39
70
7 2
7 4
76
77
79
81
82
viii
Numb e r
A-9.
A-10.
B-1.
B-2.
B-3.
B-4.
B-5.
c-1.
c-2.
D-1.
E - 1 .
E-2.
E-3.
F-1.
F - 2 .
F - 3 .
F-4.
G-1.
G-2.
G-3.
H-1.
1-1.
Conversion of penicillin G into 7-aminodeacetoxycephalo- sporanic acid (7-ADCA) . . . . . . . . . . . . . . . .
Chemical structures of some semisynthetic cephalosporins
Structures of many of the cephalosporin antibiotics . .
Biosynthetic pathway for the formation of cephalosporin
Chemical deacylation of Cephalosporin C . . . . . . . .
PaRe
84
85
9 2
. 93
96
Schematics for recovery and concentration of cephalosporin C 100
Cephalosporin C purification from broth by adsorption . . 102
Production scheme for oxytetracycline . . . . . . . . . . 106
Production chart for chlortetracycline . . . . . . . . . 107
Therapeutically important sulfa drugs and sulfones . . . 114
Bacitracin production flow and equipment scheme . . . . . 120
Block diagram for the production of bacitracin . . . . . 121
HPLC chromatogram of purified bacitracin . . . . . . . . 124
Common stirred-tank batch fermenter for ethanol production 128
Equipment used in the production of ethanol from corn stover . . . . . . . . . . . . . . . . . . . . . . . . . 129
Purification equipment for 95% ethanol distillation . . . 130
Purification equipment for anhydrous ethanol production . 1 3 1
Intracellular enzyme purification f l o w sheet . . . . . . 136
Alternate enzyme recovery processes . . . . . . . . . . . 137
Common commercial enzyme process . . . . . . . . . . . . 138
Block diagram of the Hutchinson, MN, SCP production facility . . . . . . . . . . . , . . . . . . . . . . . . 142
The effect of drying temperature on survival of freeze driedcells. . . . . . . . . . . . . . . . . . . . . . . 149
ix
Number Pane
J.l . Piping diagram for fermenter air filtration . . . . . . . 154
5.2 . Survival curves for sterilization of spores . . . . . . . 154
5.3 . Continuous steam sterilization of medium feed . . . . . . 157
5.4 . Continuous sterilization using heat exchangers . . . . . . 158
K.1 . Standard fermenter diagram . . . . . . . . . . . . . . . . 164
K.2 . Typical piping arrangement for sampling and inoculation . 1 6 5
K.3 . Geometric configuration for agitator placement in a typical fermenter . . . . . . . . . . . . . . . . . . 166
K.4 . Agitator shaft seal for a fermenter . . . . . . . . . . . 167
K.5 . Mechanical shaft seal for fermenter agitator . . . . . . . 168
L.l . Schematic diagram showing the many types of chromatography that are in use . . . . . . . . . . . . . . 174
L.2 . Process chromatograph flow diagram . . . . . . . . . . . . 1 7 5
L.3 . Sample chromatograms for the separation of cephalosporin C 176
L.4 . Schematic diagram of continuous rotating annular chromatograph . . . . . . . . . . . . . . . . . . . . . . 178
L.5 . Schematic diagram for supercritical fluid extraction using GO2 . . . . . . . . . . . . . . . . . . . 181
xi
EXECUTIVE SUMMARY
OBJECTIVE
It is desired to determine the most efficient methods for
meeting the antibiotic needs of the population for the period
beginning 30 days after a nuclear disaster.
BACKGROUND
Meaningful preparedness for catastrophic disasters which would
cause mass casualties includes both development of capabilities for
reducing the impact of the hazard, and steps to deal with the
effects that cannot be prevented. A catastrophic disaster could
destroy production capabilities for antibiotics and create, at the
same time, conditions under which epidemic diseases and large
numbers of injuries occur. The availability of broad-spectrum anti-
biotics could be of major importance to the survival and recovery of
the threatened population. One purpose of this project is to
identify several antibiotics for emergency production based on
clinical need and efficiency of production and provide documen-
tation on the production of the antibiotics. We also propose
modification of antibiotic production equipment, material, and
processes that would allow expedient production of broad-spectrum
antibiotics under the austere conditions following a nuclear attack.
PROCEDURE
The approach used to obtain the necessary information and
develop the desired instructions for the expedient production of
antibiotics involve the following:
1. Select antibiotics for emergency production, based on need
and production capabilities.
2 .
3 .
4 .
5 .
6 .
xii
Document current production technologies including equipment,
materials, and processing steps for the selected antibiotics.
Document current U.S. antibiotic production sites, potential
capacities, and procedures for expedient contacting of plant
managers after a nuclear attack.
Identify potential emergency production sites which lie outside
of the risk areas, making recommendations as to how these
facilities could be used for production of antibiotics.
Recommend modifications in current production technology and
functional substitutions for certain basic equipment and facil-
ities which would allow rapid conversion of emergency produc-
tion sites to antibiotic manufacturing.
Identify alternative sources of antibiotics.
CONCLUSIONS
1. Antibiotics chosen are penicillin V and amoxicillin (from peni-
cillin G)(and combined with clavulanic acid, an alternative if
cephalexin is not able to be produced), cephalexin (a cephalo-
sporin), tetracycline and oxytetracycline (broad-spectrum,
widely tolerated, and widely prescribed and available),
bacitracin (as a topical antibiotic), and sulfanilamides (sul-
famethazine, sulfamethizole, sulfathiazole, etc.) which are
chemically produced.
The expedient dosage form should be oral. Capsule, tablet, and
suspension forms provide enhanced storage stability and de-
creased need for sterility, purity, and quality control
required for injectable forms. Fewer and simpler separation
and purification methods can be used. Transportation is
simplified and dosage control for oral antibiotics is feasible
outside of sterile hospital locations.
Several large antibiotic-manufacturing facilities lie outside
of the risk areas and probably will not be damaged.
xiii
4 . Several large antibiotic-manufacturing facilities lie just
within the risk areas and may sustain only minor damage.
5. Pharmaceutical companies often maintain a 4 - to 16-week inven-
tory of finished products and a 4 - to 8-week working inventory
of finished products ready for formulation. Distribution facil-
ities for these companies located in major cities throughout
the country also may have substantial inventories of
antibiotics. Company headquarters should be contacted to
determine the status of warehoused antibiotics. Boston,
Charlotte, Miami, St. Louis, Louisville, Kansas City, Seattle,
San Francisco, Chicago, Dallas, and Atlanta have major distri-
bution facilities.
6 . All existing antibiotic-manufacturing facilities should be
surveyed immediately after an attack.
a. Undamaged facilities should be operated at maximum capacity
for a selected antibiotic, subject to feedstock and solvent
availability.
b. Rapid assessment should be made of damaged facilities to
determine repair feasibility.
7 . Design protocols for the conversion of undamaged non-antibiotic
production sites to antibiotic production must be determined.
A "model" retrofit design to an existing plant (e.g., penicil-
lin V production at the Hutchinson, MN, SCP plant) should be
made.
a. Provide State and Federal personnel with very accurate
resource estimates of time, manpower, and materials
required.
The outcome of this model design would be:
b. Establish methods of planning for conversion.
c. Evaluate and determine the best separation method for
retrofit under austere conditions.
d. Provide State and Federal personnel with a retrofit design
at once-could also serve as the model for the other
retrofits which might be needed.
8. Designated possible expedient antibiotic-production sites
9
10
11
12
xiv
should be surveyed, and their management should be informed of
impending conversion to antibiotic production.
Personnel who are familiar with a chosen emergency antibiotic-
production plant must be brought together. Included are the
operating engineer, plant operators, and chemical and
biochemical engineers. A l s o someone who is familiar with the
biological needs of the fermentation organisms and the growth
and fermentation of microorganisms (such as a biochemist,
molecular biologist, microbiologist, etc.) is necessary.
Someone with plant construction or modification experience is
also desirable to help plan the modifications and implement the
des ired changes.
To assure the availability of personnel who are adequately
trained in the use of the equipment and the process, con-
sideration should be given to training military personnel who
have a background in this area. A listing of technical experts
both within and outside of the military should be assembled and
kept current.
Antibiotic production couples a relatively small, highly
specialized, aerobic fermentation section with a complex
multistep separation/purification section. Expedient produc-
tion at alternate sites will only be feasible if there is a
significant simplification in the separation and purification
sections of the process. This may be accomplished by reducing
the number and complexity of the separation steps by using new
separation methods. Other methods that produce a usable product
in one or two steps, such as filtration followed by spray dry-
ing, may also be used, thereby producing an impure product.
Impure products are generally acceptable only for oral use.
Primary alternate sites must include an in-place aerobic fer-
mentation system and should contain some existing separation/
purification equipment which could be adapted to one of the
antibiotic production schemes.
. The specialized microorganisms used in the various antibiotic
fermentations must be procured and kept viable. Many strains
xv
of an organism can be used to produce a given antibiotic, but,
if the most efficient strain is not selected and used, plant
capacity can be substantially reduced (by ten to one hundred
times). The desired strains of the organisms should be stock-
piled by freeze drying or alternate long- term storage techni-
ques that would allow the cultures to be immediately available
to expedient producers. Consideration should also be given to
stockpiling some specialized equipment, finished bulk chemicals
(sulfur, 6-APA, etc.), and other critical supplies.
13. Small volume antibiotics with specialized uses (e.g., lincomy-
cin for serious bacterial infections in penicillin-allergic pa-
tients) should be stockpiled, and responsible funding agencies
should be identified.
14. Many veterinary antibiotics are identical to human drugs, but
lower in purity. They could be used with caution in an emer-
gency.
15. Substantial antibiotic capacity is available in Mexico and
Puerto Rico. Consideration should be given to importation of
the necessary antibiotics from these locations.
16. The raw materials necessary for fermentations are generally
available. They include carbohydrates (such as glucose,
sucrose, and lactose), nitrogen sources (like corn steep
liquor or soybean meal), organic acids, and various inorganic
chemicals for pH control and growth. Separation/purification
requires organic solvents (amyl or butyl acetate, 1-butanol),
filter aid and precoats for broth filtration, and various salts
and minerals for salting-out procedures and pH control.
17. General utility requirements for antibiotic production include
plentiful steam for sterilization and for solvent recovery and
purification. Electricity is required to run the pumps, cen-
trifuges, extractors, mixers, agitation equipment, and chilled
water machinery. A source of chilled water is needed to remove
heat from the fermenters and to provide cooling for the separa-
tion processes that often operate at 0 to 3°C. Basic analyt-
xvi
ical and microbiological laboratory facilities are necessary
for process control and quality assurance.
18. Several advanced separation technologies hold great promise for
making vast reductions in the separation/purification resources
required to produce purified antibiotics. These technologies
include high performance liquid chromatography (HPLC) using
continuous annular chromatography, supercritical fluid extrac-
tion, and membrane separation methods.
xvii
ABSTRACT
The literature on the manufacture, separation and purification,
and clinical uses of antibiotics was reviewed, and a bibliography of
the pertinent material was compiled. Five antimicrobial drugs, peni-
cillin V and G (and amoxicillin with clavulanic acid), Cephalexin (a
cephalosporin), tetracycline and oxytetracycline, Bacitracin (topi-
cal), and sulfonamide (chemically produced) were identified for
emergency production. Plants that manufacture antibiotics in the
continental United States, Mexico, and Puerto Rico have been iden-
tified along with potential alternate sites such as those where SCP,
enzyme, and fermentation ethanol are produced. Detailed process flow
sheets and process descriptions have been derived from the litera-
ture and documented.
This investigation revealed that a typical antibiotic-manufact-
uring facility is composed of two main sections; (1) a highly special-
ized, but generic, fermentation unit and (2) a multistep, complex
separation and purification unit which is specific to a particular
antibiotic product. The fermentation section requires specialized
equipment for operation in a sterile environment which is not usually
available in other industries. The emergency production of antibi-
otics under austere conditions will be feasible only if a substantial
reduction in the complexity and degree of separation and purity nor-
mally required can be realized. Detailed instructions were devel-
oped to assist State and Federal officials who would be directing
the resumption of antibiotic production after a nuclear attack.
Initially, all plant managers should be contacted (using the lists
provided in this report) to determine if their facility has been
destroyed or damaged. If a plant has been damaged, the manager
should determine the feasibility of making repairs, the potential
capacity of the repaired plant, and the type of antibiotic that can
be produced. If enough production cannot be realized from undamaged
and slightly damaged plants, the plants from the list of alternate
emergency production sites must be contacted, determination of the
plant status made, and the managers informed of impending conversion
xvi i i
to antibiotic production. If alternate sites must be used, a team
of skilled personnel must be assembled to convert the plant to anti-
biotic production in the austere environment.
1
EXPEDIENT ANTIBIOTICS PRODUCTION
P. R. Bienkowski
C . H. Byers
D. D. Lee
1. INTRODUCTION
It is expected that the per capita demand for antibiotics in the
postattack environment will rise sharply although total demand may
decrease, remain constant, or increase, depending on the extent of
the population destroyed. This increased demand will arise from two
sources; treatment of wounds and control o f communicable diseases.
It is anticipated that a large portion of the current antibiotic
production capacity will be destroyed because most antibiotic-
manufacturing facilities lie within probable nuclear attack zones.
At the same time, problems in the control of communicable diseases
will rise sharply due to disruptions in the food and water supplies
and sewage treatment systems.
The extent to which antibiotic capacity becomes critical in a
given postattack environment depends on many factors, some of which
are interdependent. Among the variables are:
1. The relative size of population losses and antibiotic capacity
losses,
2. The antibiotic requirements per survivor relative to preattack
per capita demands, and
3 . The sizes of surviving antibiotic stockpiles.
Such determinations z r e quite cnmplex and beyond the scope of the
present study.
This report documents the clinical uses of the various an-
tibiotics and recommends antibiotics for expedient production in a
postattack environment. The current antibiotic-manufacturing sites
2
are identified along with descriptions of the manufacturing tech-
nologies for the selected antibiotics. Possible sites for producing
antibiotics under austere conditions are also identified. These
include in decreasing order of desirability: damaged but rebuildable
antibiotic production facilities; animal and plant antibiotics
producers; single cell protein producers; enzyme, msg and other
molecular biology product makers; citric acid producers; brewers and
fuel ethanol producers; the dairy industry; large hospitals and
universities with fermentation research facilities; and farm
fermentation units.
Commercial production technologies for the selected antibiotics
are documented along with recommendations on how these processes can
be modified to allow for conversion of alternate production sites to
antibiotic production. The processes for manufacturing antibiotics
are very difficult to reproduce at alternate facilities due to the
unique nature of antibiotic production. A highly specialized fermen-
tation section requiring sterile conditions is coupled to a very
large separation and purification system which requires large volumes
of solvents and a great deal of energy.
In summary, this report develops the methodologies necessary for
expanding antibiotic capacity in a post attack environment. The
extent to which these recommendations are implemented will be deter-
mined by the actual environment in existence after a nuclear attack.
By coupling this study with mathematical models which predict possible
post attack environments for assumed attacks, various possible meth-
odologies and recommendations can be explored by FEMA planners.
3
2. DEFINITION OF AN ANTIBIOTIC
Antibiotics are a class of antimicrobial chemicals which are
produced by microorganisms (mainly the saprophytic molds and bacteria
of the soil). These substances will inhibit the multiplication o f
various microorganisms or may even destroy these organisms by either
interfering with cell wall development and/or metabolism. Each anti-
biotic has its own characteristically selective "spectrum" of potency
against various microorganisms, which varies not only for the species
but also the strain of the infecting organism and other conditions.
The term "broad spectrum" is applied to antibiotics which are effec-
tive against a wide range of bacteria.' Antibiotics are in general
ineffective against typical virus diseases except for the tetra-
cyclines which are sometimes effective against large viruses. With
the exception of the penicillins and cephalosporins which are produced
by molds, all major classes of antibiotics are derived from bacteria.'
Other antimicrobial substances are produced by the higher
plants. Some are synthetically produced such as p-aminosalicylic
acid and the sulfonamides. The sulfonamides were the first major
broad spectrum antimicrobial drugs. However current antibiotics are
now more potent than the sulfonamides. The sulfonamides were origi-
nally active against a wide range of bacteria, but an increasing
incidence of resistance in bacteria formerly susceptible to the s u l -
fonamides has decreased their effectiveness (a single mutation in
the bacteria allows some organisms to develop resistance).' The
sulfonamides are still the choice medication in several common bac-
terial infections.
5
3. ANTIBIOTICS SELECTED FOR EXPEDIENT PRODUCTION
The following antimicrobial drugs have been targeted for
expedient production. They include antibiotics which are taken in
capsule or tablet or suspension form-penicillin V, the amoxicillin
derivative of penicillin G , the cephalexin derivative of cephalo-
sporin C , tetracycline and oxytetracycline, and the sulfonamides
which are produced chemically. Also selected is the topical antibiotic
bacitracin.
Penicillins, cephalexin, and tetracyclines were selected for
their widespread clinical applications because they are effective in
common respiratory and gastrointestinal tract infections and some
common opportunistic infections. Also, their share of production
capacity and dollar value means that there are many plants producing
or capable of producing them. Opportunistic infections are caused
by gram positive and gram negative (G+ and G - ) organisms such as E.
coli, some Pseudomonads and Bacteroides pathogens. They often cause
chronic infections after puncture or burn wounds and are often
resistant to penicillin but susceptible to cephalexin. A combin-
ation of clavulanic acid (a suicide inhibitor of /I-lactamase enzymes)
with amoxicillin (called augmentin) gives similar or better coverage
than cephalexin in many cases. If the production capabilities are
available (it requires the same equipment as penicillin, but uses a
different organism), it is a viable alternative to cephalexin.
Table 1 gives the 1980 and 1985 figures for antibiotic production in
the United States.3
Figures 1 and 2 show the general structures of the cephalo-
sporins, penicillins, and tetracyclines. Once a manufacturing pro-
cess is developed for one of these classes of antibiotics, many
similar antibiotics can be produced by either very minor changes in
the process (i.e., different growth media, precursors, etc.) or by
semisynthetic means. For example while there are many penicillins
in use, only three are produced naturally by fermentation (penicil-
lin G , 0, and V); all of the others are then produced from penicillin
6
Table 1. U . S . antibiotic production
Production Sales Value (1000 l b s ) 1000 lbs) ($1000) Antibiotic
Cephalosporin 977 9 7 0
Penicillin V & G 6 5 1 4 1 9 7 4 3 2 , 0 8 2 Penicillin G for medicinal 2 7 5 0 All other uses 3 7 6 4
44 8 3 2 , 2 4 6 Semisynthetic Penicillins 1 7 6 8 Amoxicillin 323 Amp ic i 11 in 9 6 4 All Others 4 8 1
Tetracyclines 6 5 6 2 4 1 2 2 8 6 , 4 8 8
Sulfonamides 4 8 4 1 7 7 1 1 0 , 8 8 4
(International Trade Commission 1 9 8 0 )
Antibiotic U.S. Production ( $ Millions) 1 9 8 5
C ephal o sp o r ins 1 , 2 1 1 .
Penicillins 2 5 2 .
Tetracyclines 2 8 0 .
All Others 7 5 4 .
TOTAL 2 , 4 9 7 .
7
ORNL DWG 87-470
COOH
CEPHALOSPORIN C
PENICILLIN G Fig. 1. Structures of Cephalosporin C and Penicillin G prodwed
in antibiotic fermentations.
8
ORNL DWG 87-471
Y OH
- OH
- CONH,
0 OH 0
- R2 R, AND R, ARE DIFFERENT IN
R l - DIFFERENT ANTIBIOTICS
H H CI H Br H H
TETRACYCLINE CHLORTETRACYCLINE BROMTETRACY CLINE OXYTETRACYCLINE OH
Fig. 2. Structure of Tetracycline showing substitutions to get related antibiotics.
9
G by enzymatic (penicillin acylase) (Fig. 3 ) and/or chemical reac-
tions. The other reaction shown in Fig. 3 is the deactivation reac-
tion which is used by penicillin resistant organisms to confer
penicillin resistance. The clinical activity of these three classes
of antibiotics covers a "broad spectrum" of uses (Table 2). Table 3
is a list of the various antibiotics and the generally recommended
dosages for a course of treatment with the antibiotics.4 Also
listed are the approximate requirements to treat a million patients
per month at the recommended treatment rate. This can be used to
estimate the total antibiotic required if the affected population
can be estimated.
Because of the possibility for a large number of burns and skin
wounds, it is desirable to have one topical antibiotic for their
treatment. Bacitracin is the most common topical antibiotic, and it
has a wide range of clinical applications.
The sulfonamides also represent a family of related drugs. The
sulfonamides were the first broad spectrum antimicrobial drugs, coming
into widespread use in the 1930s. Due to overuse, the effectiveness
of these drugs as "broad spectrum" antibacterials has been reduced
over the years as organisms built up resistance, to the point where
they do not have the same potency as the fermentation antibi0tics.l
They are still useful and the drugs of choice for urinary tract
infections (UTI) and some ear infections. Because the sulfonamides
are produced chemically and do not require the complex and special-
ized equipment necessary for the fermentation antibiotics, they
would require less production resources in a postattack environment.
3.1 PENICILLINS
Over 100 different penici ins have been produced by natural
fermentations. However, only penicillin G, penicillin V I and, to a
small extent, penicillin 0 are produced in commercial quantities.
The other natural penicillins are not therapeutically useful. Peni-
cillins act by specific inhibition of bacterial cell wall synthesis,
10
ORNL DWG 88-236
H
COOH 0
PENICILLIN G
PENICILLIN ACYLASE I
C
P H EN Y LAC ET1 C AC I D 6 -AM I N 0 P EN IC I LLA N I C AC I D
CHEMICAL R EACY LATI ON
SEMISYNTHETIC PENICILLIN
Fig. 3. Chemical modification to penicillin G to produce semisynthetic penicillins.
11
Table 2. Antibiotics produced in the United States (by classification)
ANTIBIOTIC PRODUCER ORGANISM ACTIVITY TYPE --CEPHALOSPORINS--
Cefaclor, Cefadroxil Cefamandole, Cefoxitin Cephalexin, Cefonicid, Cephaloridine, Cephalothin Cefuroxime, Cephapirin Cephacetrile, Cephradine
G+, G-, 0-Lactam 3.6,8,11 Cephalosporium skin bone,
ear, respiratory
a c r emon i um
Streptomces SP .
--SEMISYNTHETIC PENICILLINS--
Amoxicillin, Phenethicillin
Ticarcillin, Augmentin, Piperacil- 11,13 lin. Cyclacillin Epicillin. Ampicillin, Pivampicillin, Bacampicillin, Methicillin, Nafcillin, Oxacillin, Cloxacillin, Dicloxacillin, Flucoxacillin
Propicillin, Carbenicillin 4,5.9
---NATURAL PENICILLINS---
Penicillin V Penicillin G
---TETRACYCLINES-- -
G+. G- 0-Lactam Penicilli- chrvsonenum
Gt O-Lactam 4,6,8,9, Penicillin 11,13,17 chrvsonenum
Chlortetracycline, Tetracycline G+. G- Polyket ide Demeclocycline, Doxycycline 2.6.13.27 Streptomyces Rickettsia, Minocycline, Rolitetracycline SP. Mycobacteria Methacycline, Oxytetracycline Mycoplasma
---BACITRACIN--- 10 Bacillus
licheni formis B. subtilis
Acetyl Sulfisoxazole, Mafenide Acetate, Sulfabenzamide, 2,11,13, Chemical Sulfacetamide, Sulfadimethoxine 14,15 Reaction Sulfachloropyrazine, Sulfonamide, Sulfathiazole, Sulfamethizole, Sulfasalazine Sulfaquinoxaline, Sulfadiazine Sulfanitran, Sulfisoxazole Sulfamethazine, Sulfachlor- pyradi zine
Uti, Ear Sulfonamide
PRODUCING CCMPANIES
1. fbbott Laboratories 2. American Cyanamide 3. Anheuser-Busch, Inc. 4. Beecham Laboratories 5. Biocraft Laboratories. 6. Bristol-Myers Company 7. Burroughs Wellcome Co. 8. Eli Lilly and Company 9. Fennenta
10. 11. 12. 13.
Inc . 14. 15. 16. 17.
IMC Chenical Group Merck & Co., Inc. Napp Chemicals, Inc. Pfizer, Inc. Hoffmann-La Roche Inc. Salsbury Laboratories. Inc SmithKline Beckman Corp. The Upjohn Canpany
1 2
Table 3. Dosage forms and dosage recommendations for expedient antibiotics
Antibiotic DosaRe form Dosage Time Total given
Penicillin G Injection 5-15 mil. u/d 10-15d 80-375g (powdered for reconstitution)(l mil u=60Omg Pen G sodium) Oral tablets 1-3 mil. u/d 10 d 17-50g Oral syrup 0.5-3 mil u/d 10 d 8-50g
Penicillin V Oral tablets Oral syrup
0.6-2 mil u/d 10 d 0.2-2 m i l ufd 10 d
Amoxicillin Oral capsules 0.75-1.5 g/d 7-10 d Oral syrup 0.3-1.5 g / d 7-10 d
Amoxicillin + Oral tablets 0.75-1.5 g/d amox 7-10 d Clavulanic acid Oral syrup + 0.375 g/d clav. acid (Augmentin)
Cephalexin Oral capsules 1-4 g/d 10 d Oral syrup 0.5-4 g/d 10 d
Tetracycline A C l Oral capsules 1-2 g/d 7-10 d Oral syrup 0.25-2 g/d 7-10 d Inject ion 0.25-2 g/d 4-7 d
Oxytetracycline Oral capsules 1-2 g/d 10 d Oral syrup 0.5-2 g/d 10 d Injection 0.25-2 g/d 4-7 d
Sulfarnethizole or Oral tablets Sulfamethoxazole/ Oral tablets
trimethoprim Oral syrup
2-4 g/d 7-10 d 1.6gld 10-14 d 0.4-1.6 g/d 10 d
10-33g 3.3-33s
5.2-158 2.1-15g
5.2-15g 2.6-3.8g
10-40g 5-40g
7-20g 1.8-2og
1-14g
10-2og 5-2Og 1-14g
14-40g 16-22.4g 4-16g
14000 ultube Bacitracin Topical 2-5 times/d as needed
Penicillin G required per l o 6 treatedlmonth
Penicillin V required per l o 6 treatedlmonth
Amoxi c i 11 in required per 10 tr eatedlmonth
Augmentin-- Amoxicillin required per l o 6 treatedlrnonth Clavulanic acid
Cephalexin required per 10' treatedlmonth
Tetracycline required per 10' treatedlmonth
Oxytetracycline needed per l o 6 treatedlmonth
Su 1 fame th i z o le needed per 10 treated /mon th Sulfamethoxazolel
trimethoprim needed per l o 6 treatedlmonth
B a c it r ac in required per 10 treated lmon th
= 80,000 - 375,000 kg (injection) = 17,000 - 50,000 kg (tablets) = 8.000 - 50,000 kg (syrup)
= 10,000 - 33,000 kg (tablets) = 3,300 - 33,000 kg (syrup)
= 5,200 - 15,000 kg (capsules) = 2,100 - 15.000 kg (syrup)
= 5,200 - 15,000 kg (tablets) = 2,600 - 3,800 kg (tablets)
- 10,000 - 40.000 kg (capsules) = 5,000 - 40,000 kg (syrup)
= 7,000 - 20.000 kg (capsules) = 1,800 - 20,000 kg (syrup) = 1,000 - 14,000 kg (injection)
= 10.000 - 20,000 kg (capsules) = 5,000 - 20,000 kg (syrup)
1.000 - 1 4 , 0 0 0 kg (injection)
= 14,000 - 40,000 kg (tablets)
= 16,000 - 22,400 kg (tablets) = 4 , 0 0 0 - 16,000 kg (syrup)
= 14 x l o 9 units
13
and kill only growing cells, while not harming resting cells. All
three penicillins are produced by the same fermentation process with
the constitution of the growth media (the added precursor) determining
which is pr~duced.~ All of the natural penicillins are available in
dry, crystalline form and are stable for several years under control-
led conditions. Penicillin G is mainly administered parenterally in
solution form, and its stability in solution is rather limited. How-
ever, over 20 therapeutically useful semisynthetic penicillins are
derived from penicillin G .
Penicillin V, 0 , and G are produced with the submerged culture
process in 40,000- to 200,000-1 fermenters. Oxygen transfer and the
specific culture used are quite important. While many organisms can
be used to produce penicillin, the yield is strongly dependent on
the strain which is used.6,7 In general, the concentrations of
antibiotics in the fermenter are very low. However, with proper
strain selection, penicillin can be produced in concentrations of up
to 40 g/1 which is 1 or 2 orders of magnitude greater than many
other antibiotics.' Separation and purification often constitute
90% or more of the steps (especially in the production of injectable
antibiotics) in an antibiotic plant, making the concentration in the
fermentation broth very important.
3.2 CEPHALOSPORINS
Cephalosporins are economically and therapeutically as important
as the penicillins. These drugs are broad-spectrum antibiotics, active
against respiratory, ear, skin, and bone infections caused by G+ and
G - bacteria, and they have low toxicity. In a manner similar to the
penicillins, they act by inhibiting bacterial cell wall (peptidogly-
can) synthesis, and they kill growing cells but not resting cells.g
Natural cephalosporin C is the main fermentation product, but is not
very therapeutically active, and is converted to cephalexin or other
active forms which are therapeutically important. Cephalosporin C is
produced by the same fermentation process used for the penicillins
but utilizes different growth media and producer organisms. However,
final fermentation broth concentrations are much lower (1.0 to
14
4 . 0 g/L) and include many similar compounds resulting in a more
difficult separation and purification process." The active cephalo-
sporins (cephalexin or others) are normally administered orally in
capsule form.
3 . 3 TETRACYCLINES
Tetracyclines are an important class of broad-spectrum antibi-
otics that are active against both gram-positive and gram-negative
bacteria, as well as rickettsias, mycoplasmas, leptospiras, spiro-
chetes, and chlamydias. Tetracyclines act as inhibitors of protein
synthesis with the site of action being the 30s ribosome, where
binding of aminoacyl-tRNA to the ribosomal A-site is inhibited.
There are three natural and six semisynthetic tetracyclines of
economic and therapeutic importance. Like the penicillins and
cephalosporins, the tetracyclines are produced by an aerobic fermen-
tation process except that the organism is a bacteria instead of
fungi. Presently, there are about 20 different organisms which
produce tetracyclines. The yield is low, with maximum published
fermentation broth concentrations of only 2.0 g/l.l1-I3
3.4 BACITRACIN
Bacitracin is used as a topical antibiotic, and it has broad-
spectrum antibiotic effects against gram-positive and normal strep-
tococcus and staphylococcus skin infection-producing bacteria. l 4
While it is very effective when taken internally, this use is severely
restricted due to problems with toxicity. The structure of bacitracin
is shown in Fig. 4 . Bacitracin is produced by conventional aerobic
submerged culture fermentations like those of the other antibiotics.
Many different strains of bacteria can be used. The most effective
produces a fermentation broth concentration of 9 g/l. The extent to
which separation and purification control the process depends upon the
use. l 5 For animal feed uses (as a growth promoter), the separation
and purification process is fairly simple. Bacitracin is stored as
a fine white powder with good long-term stability.
15
ORNL DWG 88-282
2 CYSTEINE 3 LEUCINE
0 \ C-C / I
OH c, ,.c--C+N\ ISOLEUCINE 7 C%N ‘ \
C’C, / \SHC C / C’ 4 GLUTAMIC \
$ 5 ISOLEUCINE NH2 C’O
N,H2 0.4 \
I c-c 12 ASPASAGINE c ‘C’
C NX 0 5 c, 0 \\ %N’c-cAc,
t i
c\ /
c, F 6 LYSINE c\ 0 C C 4 w N ASPARTIC ACID $
I C-C-C-NH, I
HO\C c - c I \ O*C 7 ORNITHINE N *, x OH
1
1 1
ACID
oOL\ 0 c’ 8 ISOLEUCINE ’ c, N-#-~-~-N% I c’ \
C C C 10 HISTIDINE ,C,c /
\ LN N \
9 PHENYLALANINE C
Fig. 4. Structure of Bacitracin A. For convenience, most of the H atoms in the formula have been omitted. Source: 9. Fqdyshov, “The Bacitracins: Properties, Biosynthesis, and Fermentation,” pp 665-694 in Biotechnologv of Industrial Antibiotics ed E. J. Vandamme, Marcel Dekker, Inc., New York, 1986.
1 6
3 . 5 SULFONAMIDES
Sulfonamides are a family of chemically produced anti-infective
agents containing a benzene ring with a sulfonamido group ( - S 0 , N H - )
and an - R group in the para position. Several examples are shown in
Fig. 5. The antibacterial properties are derived from the drug’s
ability to disrupt the microorganism’s metabolism.16 Sulfonamides
are normally administered in capsule form, and they exhibit long-term
storage stability when they are stored in light-resistant containers.
Adverse effects of sulfonamides are numerous and may involve nearly
all organ systems. However, serious or fatal reactions are infre-
quent. Most adverse reactions appear to be due to hypersensitivity
and increase with increasing dosage.”
17
ORNL DWG 88-235
H /
H,N -(->%--N 0 II \ R
SU LPHAN I LAM IDE WHERE R IS:
NH2 I
= C -NH,
(5)
J-J S
(7)
H WHERE ( 1 ) IS SULFAMETHYLDIAZINE (2) IS SULFAQUANIDINE
(4) IS SULFAMETHAZINE (6) IS SULFAQUINOXALINE
(3) IS SULFATHIAZOLE (5) IS SULFATHIADIAZOLE (7) IS SULPHANllAMlDE (SULFONAMIDE)
F i g . 5. Structure of sulfonamides.
1 9
4. ANTIBIOTIC HANITFACTURERS
The first step to be taken by emergency managers after a nuclear
attack is to contact the antibiotic-manufacturing facilities for a
damage assessment. Table 4 lists all of the current manufacturing
facilities, along with their telephone numbers. As can be seen from
Fig. 6 , some of these production sites lie outside the risk areas
and may not sustain damage." The TR-82 risk areas were used in
this analysis. The (current) NAPB-90 planning base was not available
when this analysis was done. The difference should not be significant
since most antibiotic plants are located in highly industrialized
areas assumed at risk in both documents. The analysis here may be a
little more conservative (pessimistic). These include: American
Cyanamid's facility in Hannibal, Missouri; Lilly's facility in
Clinton, Indiana; Burroughs Wellcome's plant in Greenville, North
Carolina; and Salsbury Laboratories' plant in Charles City, Iowa.
Many facilities in the risk areas are near the edge and may sustain
only minor damage; for example, Eli Lilly has a very large plant
south of Lafayette, Indiana, which is at the edge of the potential
blast zone centered on West Lafayette.
20
Table 4. Risk assessment for antibiotic producers in the U. S. assuming three different attack scenarios
ANTIBIOTIC PRODUCER TELEPHONE/ MANUFACTURING LOCATION COUNTY INDUSTRIAL MILITARY ==BLAST ANTIBIOTICS
COMPANY HEADQUARTERS TELM ATTACK ATTACK +*=FALLOUT PRODUCED
==HIT ==HIT ****=BOTH
---=Clear ---=Clear ---=Clear
(TR-82)
Abbott Labora to r i e s
1400 Sheridan Rd.
North Chicago. I L 60064
800 323-9065
910 235 1584
North Chicago, I L 60064 EES _ _ _ **** Lake xxxx
American Cyanamid Company
One Cyanamid Plaza
Wayne, N J 07470
201 831-2000
130 400
Hannibal, t9J 63401 W i l l o w I s l a n d , WV 26190
Bound Brook, N J 08805 P e a r l River . NY 10965
_ _ _ Marion
P leasan t s XXXX
Somerset XXXX
Westchester XXXX
_ _ _ Tet racyc l ines
_ _ _ Tet racyc l ines
S u l f a **** **** Tet racyc l ines
Beecham Labora to r i e s
501 F i f t h S t .
B r i s t o l , Tenn. 37620
615 764-5141
201 469-5200
B r i s t o l , TN 37620
Piscataway. N J 08854
S u l l i v a n XXXX Somerset XXXX
xxxx Semi-synthetic **** P e n i c i l l i n s
B i o c r a f t Labora to r i e s , Inc 92 Route 46 Elmwood Park, N J 07407
201 796-3434 Waldwick. N J 07463 Bergen XXXX xxxx Semi - syn the t i c
Penic i l l i n s
B r i s t o 1-My e r s Company
P.O. Box 4755 Syracuse, NY 13221-4755
315 432-2000 Syracuse, NY 13221 Onandaga XXXX xxxx Semi-synthetic
P e n i c i l l i n s 444 2012 i d sls
Burroughs Wellcome Co.
3030 Cornwall is Road Research Tr i ang le Park N.C. 27709
919 248-3000 Greenv i l l e . NC 27834 P i t t _ _ _ Acyclovir , _ _ _ t r imethopr i m
E l i L i l l y and Company
307 E . McCarty
Ind ianapo l i s , I N D . 46206
317 261-2000 Ind ianapo l i s , I N 46285 Cl in ton , I N 47842
317 474-1430 Lafaye t t e . I N 47902
Marion XXXX
Vermil l ion --- Tippecanoe ---
_ _ _ **** P e n i c i l l i n V ,
_ _ _ Cephalosporins
-__ xxxx Cephalosporins
_-_
Fermenta
510 E . Union S t .
P.O. Box 1808 West Ches te r , PA 19380
215 436-7500 West Ches t e r , PA 19832
902517
Chester XXXX P e n i c i l l i n V . G fi
Semi-Synthetic
P e n i c i l l i n s
_ _ _ _ _ _
312 296-0600 Ter re Haute, I N 47808 Vigo xxxx xxxx B a c i t r a c i n _ _ _ IK Chemical Group
666 Garland P lace
Des P l a i n e s , I L 60016
2 1
Table 4. (Continued)
ANTIBIOTIC PRODUCER TELEPHONE/ MANUFACTURING E A T I O N COUNTY INDUSTRIAL MILITARY XXXX=BLAST ANTIBIOTICS
COMPANY HEADQUARTERS TELEX ATTACK ATTACK +*=FALLOUT PRODUCED
XXXX=HIT ==HIT ****=BOTH
---=Clear ---=Clear ---=Clear
Merck h Co., Inc . 126 E . Lincoln Ave.
Rahway, N 3 07065
Napp Chemicals, Inc
199-T Main S t . Lodi, N3 07644
P f i z e r Inc.
235 E . 42nd S t .
New York. NY 10017
Hoffmam-La Roche Inc . 340 Kingsland S t .
Nutley, New Je r sey 07110
Salsbury Labora to r i e s , Inc
2000 Rockford Rd.
Charles C i t y , I A 50616
SmithKline Beckman Corp.
1500 Spring Garden S t r e e t
Ph i l ade lph ia , PA 13101
E . R . Squibb & Sons
P . O . Box 4000 Princeton. N . J . 08540
The Upjohn Company 7000 Portage Road
Kalamazoo, Michigan 49001
201 574-4000 Albany, GA 31705 Dougherty XxXx xxxx Su I f a , Thienn lmy c i n --_ --- Cephalexin
r i b o f l a v i n
Elkton, VA 22827 Rockingham --- -__ --_ -__ Danv i l l e , PA 17821 Montour ---
314 353-7000 St. Louis, Mo 63116 S t . Louis XXXX xxxx )(xxy none
201 773-3900 Lodi, N J 07644 Bergen xxxx xxxx S u l f a 134 649
--_
--- 212 573-2323 Groton. CT 06340 New London XXXX --- Ter re Haute, I N 47808 Vigo xxxx
Milwaukee, W I 53212 Milwaukee )(xxx ---
--- 201 235-5000 Belvidere , N J 07823 Warren xxxx 133 417 Nutley, N J 07110 Essex xxxx ---
800 247-1833 Charles C i t y , I A 50616 Floyd ---
215 854-4000 Conshohocken, PA 19428 Montgomery --- 83 4487
609 921-4000 Kenly, NC 27542
616 323-4000 Kalamazoo. M I 49001 224 465
Johnston ---
Kalamazoo xx)(x ---
**** Pen, T e t r a , S t r ep to
Mw( Tet racyc l ines , e t c .
xxxx Microbial Products
S u l f a
**** Sulf a
_-_
S u l f a -- -
xxxx Cephalosporin
EES , e t c xxxx
22
ORNL D W G 87-472
Fig. 6. Antibiotic producer locations superimposed on U. S. map with two types of nuclear attacks.
23
5. ANTIBIOTIC PRODUCTION TECHNOUXIES - GENERAL DESCRIPTION
Antibiotic-manufacturing plants contain two main processing
segments; a highly specialized fermentation section and a large,
energy-intensive separation and purification section. While the
fermentation section generally occupies about 50% of the space and
accounts for about 50% of the equipment costs, it accounts for only
a relatively small portion of the processing steps required for
antibiotic production (approximately 10% of total steps are fementa-
tion related) (see Fig. 7 ) . 1 9 Highly specialized fermentation
equipment (including fermenters, air and fluid filters, agitators,
heat exchangers, pumps, etc.) is necessary. Operating conditions
specify large volumes of sterile air, a sterile environment and growth
media, low temperature cooling water or chilled water (in the penicil-
lin fermentation for example, heat removal is critical), etc. The
separation and purification section generally employs standard
equipment normally found in the chemical processing industries.
The equipment includes centrifuges and filters for separating the
microbes from the broth which contains the antibiotic. Solvent
extractors (such as the Podbielniak extractor, the first centrifugal
unit to gain commercial acceptance during its use in the separation
of penicillin) are used to separate antibiotics from the fermentation
broth. Distillation columns are used f o r solvent recovery and puri-
fication. This equipment must all be operated in a sterile manner.
These sections can contain a large number of s t e p s , similar in s i z e
and function to a small oil refinery. Figure 7 does not show the
solvent purification section which usually requires distillation
columns. The separation and purification sections use large volumes
of solvents which must, in turn, be purified and recycled, usually
by energy intensive distillation. If the antibiotic is the injectable
type, the purification is very exacting and extensive, with very
strict quality control, because the antibiotic has to be pure, with
no by-products or foreign matter. For these reasons, the orally
administered antibiotics are recommended for production during the
period which requires expedient antibiotic production techniques.
24
INCINER- 4 C A K E A T I O N
ORNL D W G 87-469R
F I L T E R AID. - F I L T R A T I O N W A S H W A T E R
I 4 I I I -
-
I
L IQUID F I R S T # S O L V E N T 4 E X T R A C T I O N W A S T E
M O T H E R LIQUOR
E X T R A C T 1
S P E N T SECOND - BUFFER 4 S O L V E N T
I S O L U T I O N E X T R A C T I O N
F I R S T \
C O N C E N - '
T R A T I O N / *
v - THIRD LIQ 'ID C- E x TR A C T I 0 N W A S T E
I
- S E C O N D F I R S T
C O N C E N - - C R Y S T A L - S E C O N D
C R Y S T A L - C- LIZATION L l Z A T l O N T R A T I O N
< F I R S T
C R Y S T A L S
A C T I V E D I S S O L V E
/ S O L V E N T DEcOLORING
S E C O N D C R Y S T A L S \ P A N D C A R B O N AND 4
1
\ * I
\ * J
>
FERMENTATION PROCESS
EQUIPMENT
P U R E C R Y S T A L S
F i g . 7 . Block diagram for a general antibiotic production process.
- T H I R D M O T H E R L I Q U O R C R Y S T A L - --t , V A C U U M
P R O D U C T D R Y I N G L IZATION -
O F T H I R D C R Y S T A L L I Z A T I O N
2 5
5.1 R A W MATERIALS AND UTILITIES
The most important raw materials are the organisms themselves,
because although many different organisms can produce a particular
antibiotic, the yield and final fermentation broth concentration are
strongly dependent on the particular organism used. For example, at
40 g/liter broth penicillin concentration, 50,000 kg of penicillin
would require about 6 . 3 fermentation batches of 200,000 liters per
batch.5 If the concentration were only 4 g/liter (still high compared
to the wild strains), 6 3 fermentation batches would be required,
greatly increasing the separation and purification, energy, and raw
material requirements. The time necessary would also increase pro-
portionately. The most efficient organisms for each type of anti-
biotic are discussed in the appropriate appendices.
The energy and carbon sources required by the organisms along
with other nutrients are provided by the growth media. The antibiotic
precursors, such as phenylacetic acid for penicillin, are also
important in order to produce the desired type of antibiotic. The
growth medium is quite important and specific to the particular
organism used and the antibiotic desired. Table 5 lists the com-
ponents of a typical growth media for penicillin in a shake flask
or factory. Growth media for the other types of antibiotics are
given in the appropriate appendices.
Most antibiotics are separated by filtration from the fermen-
tation broth and further purified by solvent extraction which requires
organic solvents, acids, and bases. The specific chemicals required
are dependent on the particular antibiotic being produced, the
desired purity, and the end use (whether formulated for oral delivery
or for injection) (see appropriate appendices).
Utility requirements consist of sterilized air, purified water,
cooling and chilled water, electricity, and steam. The exact
C O ~ S L L T ~ ~ ~ O ~ depends dramatically on the particular antibiotic which
is being produced and is covered in the appropriate appendices.
Large amounts of cooling and chilled water are required to control
the large amount of heat evolved during aerobic fermentations and
26
Table 5. Growth media for penicillin - shake flask and factorya
Corn-steep Calam and Component liquor medium Hockenhull's
medium
Main carbohydrate- shake-flask-- lactose 3 . 0 - 4 . 0 production-- continuous glucose
3 . 0
Other carbohydrates Glucose 0.0-0.5 1.0 Starch 1.5 Non-reducing polysaccharides variable
Specific precursors Phenethylamine and
Phenylacetic acid Continuous supply for production 0.05 other precursors variable
Organic acids Acetic acid Citric acid Lactic acid
0.05
0.5
0.25 1 . 0
Main nitrogen source Amino acids, peptides, amines Variable Ammonium sulphate 0.5
Other nitrogen sources Ammonia Ethylamine
Total nitrogen Total solids
Variable 0 . 3
0.15-0.2 0 . 2 8 . 0 - 9 . 0 8.5
aD. Perlman, "Chemically defined media for antibiotic produc- tion," Ann. N. Y. Acad. Sci. 139 258-269 ( 1 9 6 6 ) .
27
because the optimum fermentation temperature is usually near ambient.
Consumption of electricity is high due to the large amount of agita-
tion required for oxygen transfer in the aerobic fermentations (5 to
40 hp/1000 gal) and for the production of chilled water. For those
antibiotics which are recovered by solvent extraction, large steam
consumptions result from the purification of the extracting solvents
by distillation for recycle back to the purification section.
5 . 2 FERMENTATION
Operating an antibiotic fermentation unit requires that special
consideration be given to culture preservation, growth media, air
and media sterilization, oxygen transfer, and temperature control in
the fermenter. Equipment that is capable of being sterilized and
maintained in aseptic condition during operation is also essential.
Many different cultures can be used to produce a specific anti-
biotic; however, the fermenter yield (units of antibiotic per unit
fermenter volume), which helps determine plant capacity, varies dra-
matically with the specific culture used. Long-term preservation of
an efficient culture is vital to antibiotic manufacturing. Three
methods used for culture preservation: subcultivation, low-temper-
ature storage, and freeze drying (see Appendix I)." Each company
jealously guards its own production organisms as trade secrets.
The growth medium is an important part of an antibiotic fermen-
tation, influencing the yield and, in some cases, the specific anti-
biotic which is produced (penicillin G, V, or 0 depending on the
precursor ~upplied).~ The growth medium can be rather complex and
is different for each antibiotic produced. See Table 5 for a typical
growth medium for penicillin.
Antibiotics can be destroyed or inactivated by some microor-
ganisms, and/or microorganisms can invade the fermentation broth and
disrupt the pure culture fermentation. In both cases a substantial
reduction in yield will result. To prevent contamination, a sterile
environment must be maintained. Fermentations require large volumes
of air which must be sterilized to remove bacteria which have particle
sizes down to 0.5 /I. There are three commercial methods for air
sterilization: (1) heat treatment (whole air supply maintained at
2100 " C ) , (2) filtration through fibrous media and filtration through
granular media (see appendix J).
In order to maintain an aseptic environment, specialized equip-
ment is required for the fermenter, transfer pumps, etc, that can be
easily cleaned and sterilized. The culture must be increased in
size several times before a commercial fermenter can be charged (see
Fig. 8 ) . " Each one of these steps requires a sterile transfer.
Steam and chemical sterilization methods are used for in-place
sterilization of equipment." Appendices J and K cover specialized
equipment and sterilization methods. Antibiotic fermentations are
aerobic with optimum operating temperatures in the 20 to 30°C range.
Oxygen transfer in the fermentation broth can be a controlling vari-
able with vigorous agitation required. Aerobic fermentations generate
a great deal of heat which results in demands for chilled water for
cooling. In addition, the vigorous agitation produces foaming pro-
blems, which often cannot be dealt with using standard antifoam
chemicals because they cause downstream separation problems.23
5.3 SEPARATION AND PURIFICATION
All antibiotic fermentations are generic in nature (i.e., they
can all use the same equipment). Different antibiotics are produced
by using different cultures and growth media, while some alterations
in operating conditions are required for optimum performance. The
separation and purification sections of an antibiotic plant can dif-
fer substantially, depending on the antibiotic which is being pro-
duced. The specific separations required for the antibiotics selected
for expedient production are delineated in the appropriate appendices.
In general, filtration, centrifugation, extraction, adsorption, and
crystallization are involved. Some veterinary antibiotic are produced
by spray drying the filtration supernatant without the usual extensive
29
PHENYLACETIC 3 ACID
--
c
03 C3
ORNL DWG aa-253
FILTER
5 FILTRATE
LYOPHILIZED INOCULUM SPORES CULTURES -
Fig. 8 . fermentation.
C
PRODUCTION FERMENTER
I CARBON, NITROGEN
PREFERMENTERS MYCELIUM
'/ PURIFICATION
30
purification steps required for human antibiotics.22.24 Under
austere conditions, with reduced purity requirements, spray drying
may be an attractive alternative allowing production of a dry product
for oral use (impure products cannot be used for injection). Two
recent developments in separation technology have the potential to
substantially reduce the size of the separation/purification section
of antibiotic-manufacturing plants. These are high performance
liquid chromatography and supercritical fluid extraction (see Appendix
L). These new methods may become increasingly important in the next
three to five years as single-step, modular units which can separate
specific antibiotics easily and inexpensively.
5.4 ANALYTIC PROCEDURES/QUALITY CONTROL
Chromatography and fluorescence detection are standard methods
for assaying antibiotics. High-performance liquid chromatography
(HPLC) uses regular and reverse phase and ion exchange packings,
with ultraviolet light as the detector source, to analyze for the
penicillins and cephalosporins, and recently for the tetracy-
clines.25g26 Thin-layer paper and gel chromatography are used for
bacitracin and the tetracyclines.2’ HPLC assays are particularly
important for the B-Lactam antibiotics in order to detect impurities
from degradation products and intermediates which are quite similar
to the active antibiotic and result in toxic reaction in humans.28.29
When given in clinical dosage, the major toxic effect of the pure
antibiotics is an allergic reaction and is quite rare. The major
toxic reaction from impure antibiotics is an allergic reaction to
the intermediates and degradation products and is much more common.
5.5 ADVANCED TECHNOLOGIES
Three relatively new and potentially very powerful separa-
tion/purification methods for antibiotics are (1) chromatography
(particularly continuous annular chromatography), (2) supercritical
fluid (SCF) extraction and ( 3 ) r n e m b r a n e ~ . ~ ~ - ~ ~ If the size of the
antibiotic-manufacturing process is to be substantially reduced,
this reduction will have to come in the separation and purification
3 1
sections of the plant which represent well over half o f the total
plant size. Chromatography, SCF extraction, and membrane tech-
nologies are relatively new in regard to their application to the
antibiotic purification process. Most of this work is still in the
developmental stages, although some penicillins and cephalosporins
are separated commercially using HPLC and/or membranes. j9' 4 0 Several
European patents have been filed on the use of SCF extraction for
antibiotic separation and purification.41 These new technologies
not only have the potential for a vast reduction in the size of the
separation equipment, but also in the total number of processing
steps. Normal antibiotic separations require between 40 and 60
processing steps. The new technologies offer much greater selectivity
which should translate into a substantial reduction in the number of
processing steps required.
While these new technologies could not be used to retrofit an
antibiotic plant if a national disaster occurred in the near future,
they will greatly simplify such a conversion within the next 3 to 5
years and beyond. It would be possible to make good estimates of
the impact of either annular chromatography or SCF extraction on the
antibiotic process in a retrofit situation because most of the
pertinent information is available in the literature. These new
separation technologies could have a similar impact on the separation
and purification of antibiotics that submerged culture fermentation
had on the fermentation end of the process. In the 1 9 4 0 s , antibiotic
plants were very inefficient because surface culture fermentations
were used.5 When, in 1943, the submerged culture process was demon-
strated at the USDA Peoria Labs, one 500-gallon fermenter using the
submerged culture process outproduced all of the large surface-
culture antibiotic plants that were operating in England.5
3 3
6 . EXPEDIENT PRODUCTION OF ANTIBIOTICS
Based on the TR-82," Industrial Emphasis and Military Emphasis
attacks, it is anticipated that insufficient antibiotic-production
capacity will remain intact to meet the needs of the population. The
first expedient production measure to be taken should be a damage
assessment of existing antibiotic-manufacturing facilities and a
determination of feasibility o f timely repair. If antibiotic-
production capacity is still insufficient, alternative facilities
will have to be converted to antibiotic production.
6.1 ALTERNATE PRODUCTION SITES
The most promising candidates for conversion to antibiotic pro-
duction are animal or plant antibiotic producers, enzyme or molecular
biology product production plants, single-cell protein producers, and
fermentation ethanol producers. Tables 6 and 7 list the locations
and telephone numbers of these plants.42 Figures 9 and 10 illustrate
the proximity of these plants to the nuclear attack zones. Some
possible candidates for conversion are listed in Table 8. These
include enzyme, molecular biology product (mono sodium glutamate
(MSG), vitamin B - 2 and B-12, xanthan, and other polymers) makers,
single cell protein producers, citric acid producers, and breweries.
Large hospitals with research facilities, universities, mushroom
producers, the dairy industry, and farm ethanol fermentation units
may provide additional candidates.
6.2 CONVERSION TO ANTIBIOTIC PRODUCTION
Antibiotic production is a two-step process: (1) production of
the antibiotic via fermentation of the raw materials in the growth
media, and (2) separation of the antibiotic from the fermentati.on
broth and purification to an acceptable level for the required end
use. The fermentation section of the process is quite specialized,
and would be very difficult to retro-fit to a plant without fermen-
tation capability. For this reason, a plant which is to be converted
34
Table 6. Risk assessment for enzyme producers in the U.S. assuming three different attack scenarios
E N N Z P E PRODUCER TELEPHONE/ MANUFACTURING LOCATION COUNTY INDUSTRIAL MILITARY XXM(=BLAST
COMPANY HEADQUARTERS TELEX ATTACK ATTACK +++=FALLOUT ==HIT XXXX=HIT ****=BOTH
---=Clear ---=Clear - - - ;Clear (TR-82)
Amber Labora tor ies 433 E. Michigan S t . Milwaukee. W I 53202
Miles Labora to r i e s , Inc .
P.O. Box 932
Elkha r t , I N 46515
Norwich Eaton Pharmaceut
17 Eaton Ave. Norwich. NY 13815
U. S . Biochem.Corp. P . O . Box 22400
Cleveland. OH 44122
_- - -__ - -_ 414 271-6755 Juneau, W I 53039 Dodge
219 264-8111 C l i f t o n , N J 07015 Madison, W I 53701
607 335-2111 Norwich, NY 13815
216 765-5000 Cleveland, OH 44128
980718
Pharmacia P-L Biochem.,Inc. 414 225-2600 Milwaukee, W I 53202 2202 N . B a r t l e t t Ave.
Milwaukee. W I 53202
Novo Labora to r i e s , Inc . 203 762-2401 Wilton, CT 06897 59 Danbury Road 964 385
Wilton, CT 06897
Gist-Brocades USA Inc . 704-527-9000 Kings t ree , SC 29556 Po Box 241068
C h a r l o t t e , NC 28224
Grain Processing Corp. 319 264-4211 Muscatine. I A 52761
1600 Oregon St 46 8497
Muscatine, I A 52761
FT4P Fermentation P r o d . , I n c . 414 347-7464 Peoria, Ill. 61650 709 N.E. Water St.
Peor i a , I l l . 61650
Merck Chern. Mfg. Div. 201-574-4000 Danvi l le , PA 17821 Po Box 196
Danvi l le , PA 17821
_-_ Passaic XXXX
Dane xxxx _-_
_-_ Chenango - - -
Cuyahoga XXXX ---
_ _ _ Milwaukee XXXX
F a i r f i e l d XXXX _ _ _
W i l l iamsburg -- -
Muscatine - - -
-_ - Peoria xxxx
Montour ---
**** xxxx
****
xxxx
35
Table 6 . (Continued)
ENNZYME PRODUCER TELEPHONE/ MANUFACTLlRING LOCATION COUNTY INDUSTRIAL MILITARY ==BLAST
CCMPANY HEADQUARTERS TELEX ATTACK ATTACK +++=FALLOUT XXXX=HIT XXXX=HIT ****=BOTH ---=Clear ---=€lear ---=Clear
_ _ _ **** Atomergic Chemetals Corp.516 349-8800 Plainview. NY 11803 Nassau XXXX 100 F a i r c h i l d Ave. 14 4612
Plainview. NY 11803
*+ SmithKline Beckman Corp. 714-871-4848 Carlsbad. C a l i f . 92008 San Diego XXXX _ _ _ 6200 El Camino Real
Car l sbad , C a l i f . 92008
XXXX - -_ The Upjohn Co . 616 323-4000 Kalamaroo, M I 49001 Kalamazoo XXXX 7000 Portage Road 224 465
Kalamazoo, M I 49001
Revlon, I n c . 212-572-5000 Kankakee, I L 60901 _ _ _ _ _ _ Kankakee --- PO Box 511
Kankakee. I L 60901
- -_ +*** Accurate Chem.&Sci.Corp. 516 433-4900 Westbury, NY 11590 Nassau xxxx 300 Shames Drive 800 645-6264
Westbury, NY 11590 14 4617
CFC I n t e r n a t ' l Inc. 201-894-4000 Argo, I L 60501 PO Box 347
Argo, I1 60501
_ _ _ **** American Hoechst Cow. 619 450-9600 La J o l l a , CA 92037 San Diego XXXX 10933 Torry Pine RD. La J o l l a . CA 92037
xxxx _ _ _ P f i z e r Inc. 414 332-3545 Milwaukee, W I 53212 Milwaukee XXXX
4253 N. P o r t Washington Ave. Milwaukee, W I 53212
Chem. Dynamics Corp. 201 753-5000 S . P l a i n f i e l d , N J 07080 Somerset )(xxx _ -_ **** 3001 Hadley Road 003 447
South P l a i n f i e l d , N J 07080
Anheuser-Busch, I n c . 314 577-2000 S t . Louis, K, 63118 S a i n t Louis XIOM XXXX xxxx One Busch Place
S t . Louis, M3 63118
36
Table 7 . Risk assessment for fermentation ethanol producers i n the U.S. assuming three different attack scenarios
FERMENTATION ETHANOL TELEPHONE/ MANUFACTURING LOCATION COUNTY INDUSTRIAL MILITARY XXXX=BLAST Capaci ty
COMPANY HEADQUARTERS TELEX ATTACK ATTACK +++=FALLOUT ( m i l l i o n
==HIT ==HIT ****=BOTH g a l i y r ) ---=Clear ---=Clear ---=Clear
(TR-82)
Archer Daniels Midland Co.
FO Box 1470
Decatur, I L 62525
Georgia- Pacif i c Corp
133 Peachtree S t . NE
At lan ta . GA 30303
Grain Processing Corp
1600 Oregon S t Muscatine. I A 52761
Midwest So lven t s Co. I n c .
1300 Main S t Atchison. KS 66002
xxxx xxxx )220
xxxx
--- 319 398-0600 Cedar Rapids, I A 52413 Linn xxxx
309 673-7828 Peor i a , I L 61600 Peoria xxxx _-- --_ 217 424-5200 Decatur, I L 62500 Macon
_ _ _
6 --_ _ - _ 404 521-4000 Bellingham. WA 98225 Whatcorn ---
EO h s c a t i n e --- --- - -_ 319 264-4211 Muscatine, I A 52761 46 8497
22 913 367-1480 Atchison, K S 66002 Atchison --- - -_ --_ 309 353-3990 Pekin, I L 61554 Tazewell XXXX xxxx 9 - -_
a Nabisco Brands, Inc.(CCFC) 319 242-1141 C l in ton , I A 52732 C l in ton XXXX _ _ - --- 600 Three F i r s t Nat ional Plaza
Chicago, I L 60602 312 269-5300
Pekin Energy Co
Po Box 10
Pekin, I L 61554
_ - _ 309 346-1121 Pekin. I L 61554 Tazewell )cxxx
A. E . S t a l e y MFG. Co. 217 423-4411 Loudon, TN 37774
2200 Eas t Eldorado S t Decatur, I L 62525 Decatur. I L 62525
Amber Labora to r i e s
4 3 3 E . Michigan S t Milwaukee, W I 5320
4 1 4 271-6755 Juneau, h'I 53039
xxxx EO
XxxX 60 South Po in t Ethanol Inc . South Po in t , OH 45680 Lawrence XXXX - -_
< 10 - -_ - _ _ Alcohol Energy Carp. S t a l e y , NC 27355 Randolph ---
< l o _ _ _ - -_ -__ Baca Food and Fuel Coop. Campo, CO 81029 Baca
< 10 Bornhoft , Paul Merino. CO 80741 Logan xxxx xxxx ****
< 10 A. Smith Bowman Dist .Corp. 703 528-9091 Reston, VA 22090 F a i r f a x XXXX --_ ****
37
Table 7 . (Continued)
FERMENTATION ETHANOL TELEPHONE/ MANUFACTURING LOCATION COUNTX INDUSTRIAL MILITARY XXXX=BLAST Capaclty
COMPANY HEADQUARTERS TELEX ATTACK ATTACK +++=FALLOUT ( m i l l i o n
g a l / y r ) XXXX=HIT ==HIT ****=BOTH
---=Clear ---=Clear ---=Clear
(TI -82)
Charmel Energy Co.
Coburn E n t e r p r i s e s , Inc .
Colorado Gasohol I n c .
C r y s t a l Fue l s , I n c .
Ecological Energy, I n c .
Food and Energy, Inc .
Kentucky A g r i c u l t u r a l Energy Coop
Lenox Grain Fue l s , Inc .
Marlin Car Care
A . E. Montana. I n c .
Xaven Alcohol D i s t i l l e r y
Southern D i s t i l l e r i e s C o .
Spudcohol
Syncorp, Inc .
U. S . Gasohol Corp.
White Flame Fue l s , I n c .
Muleshoe, TX 79347
Sherman, SD 57060
Walsh, CO 81090
Bonaparte, I A 52620
Roca, NE 68430
L i t c h f i e l d . ME 04350
Frank l in , KY 42134
Lenox, I A 50851
Marlin, TX 76661
Amsterdam, MI 64723
Selma, CA 93662
Ashford, AL 36312
Pingree, I D 83262
Roberta, GA 31078
Lockeford, CA 05237
Van Buren, AR 72956
Bailey
Minnehaha
Baca
Van Buren
Lancaster
Kennebec
Simpson
Taylor
F a l l s
Bates
Fresno
Houston
Bingham
Crawford
< l o
< 10
< l o
.z 10
<lo
c 10
21
< 10
.= 10
<10
.= 10
c10
< 10
< l o
< 10 --- __- San Joaquin XXXX
.= 10 Crawford --- __- ****
38
ORNL D W G 88 -289
NUCLEAR ATTACK ON MILITARY FACILITIES
ENZYME PRODUCERS - 0
Fig. 9 . Enzyme producer locations superimposed on U.S. map with two types of nuclear attack.
39
OmNL O W G 8’ 1 1 2 9
NUCLEAR ATTACK WITH EMPHASIS ON INDUSTRIAL F A C ~ L I T ~ E S
NUCLEAR 4 T T I C K ON MILITARY FACILITIES FERMENTATlON ETHANOL PROOUCEss ~
SMALL FERMENTATWN ETHANOL
PLANTS I P R O B I B L I CLOSED) ~ 0
Fig. 10. Fermentation ethanol producer locations superimposed on U.S. map with two types of nuclear attack.
40
Table 8. Other candidates for conversion to antibiotic production
~~~ ~~~~
OTHER BIOPRODUCT MAKERS TELEPHONE/ MANUFACTURING LOCATION COUNTY INDUSTRIAL MILITARY XXXX=BLAST COMPANY HEADQUARTERS TELEX ATTACK ATTACK +++=FALLOUT
XXXX=HIT ==HIT ****=BOTH
---=Clear ---=Clear ---=Clear
(TR-82)
Acetic acid (vineKar) producers
_ _ - H. J. Heinz Co.. 412-237-5757 Pittsburgh, PA 15230 Allegheny XXXX Pittsburgh, PA 15230
Standard Brands, Inc. 212-759-4400
525 Madison Ave.
New York, NY 10022
Beatrice/Hunt-Wesson Foods 714-680-1000 Fullerton, CA 92643
1645-T W. Valencia Dr.
Fullerton, CA 92643
Citric acid producers
Miles Laboratories 219-262-7453 Elkhart, IN 46515
Citro-Tech Products
Po Box 932
Elkhart, IN 46515
Mayo Chemical Co.
5 5 4 4 Oakdale Rd.. S.E.
Srnyrna, GA 30080
404-696-6711 Snyrna, GA 30080
American International Chemical, Inc. Natick, MA 01760 209 W. Central St. 617-655-5805
N a t i c k . E24 01760
Eartlett Chemicals Inc. 504-734-1971 Jefferson, LA 70181 Po Drawer 10710
Jefferson. LA 70181
- -_ Orange XxxX
Elkhart ---
Cobb
_ _ - Middlesex XXXX
Jefferson XXXX -_ -
****
****
XXXX
+++
xxxx
Mallinckrodt, Inc. 800-325-7155 St. Louis, Mo 63147 St. Louis XXXX XxxX xxa 2nd & Mallinckrodt sts. 800-325-8888
St. Louis. MO 63147
_-_ **** Gallard-Schlesinger Chemical Mfg. Corp. Carle Place, NY 11514 Nassau xxxx 7 8 4 Minenla Ave 516-335-5600
Carle Place, NY 11514
Pfizer Chemical Div. Pfizer, Inc. Groton, CT 06340 New London XXXX _ -_ 235-T E. 42nd St. 212-573-2323 Terre Haute, IN 47808 Vigo xxxx New York, NY 10017 Brooklyn, NY Kings x)(xx
--- --- _ -_ Southport. NC Brunswick )(xxx
**** xxxx XXXX xxxx
41
Table 8 . (Continued)
OTHER BIOPRODUCT MAKERS TELEPHONE/ MANUFACTURING LOCATION COUNTY INDUSTRIAL MILITARY XXXX=BLAST
CCMPANY HEADQUARTERS TELEX ATTACK ATTACK *=FALLOUT ==HIT ==HIT ****=BOTH
---=Clear ---=Clear ---=Clear
(TR-82)
Gluconic and Klutamic a c i d producers
PMP Fermentation P r o d . , I n c 414-347-7467 Milwaukee, W I 53201
PO Box 2219
Milwaukee, W I 53201
Ashland Chemical C o . 614-889-3333 Columbus, OH 43216 PO Box 2219
Columhus, OH 43216
Napp Chemicals, I n c . 201-773-3900 Lodi. N J 07644 199 Main S t .
Lodi, N J 07644
S t a u f f e r Chemical C o . 216-831-0200 Cleveland, OH 44122 Cowles Chem. Div.
3737-T Park Eas t Dr. Cleveland, OH 44122
S t a u f f e r Chemical Co. 203-222-3000 Westport , CT 06881 Food Ing red ien t s
Nyala Farm Road Westport . CT 06881
Vitamin B-2 o r B-12 producers
P f i z e r Inc . 212-573-2323 Milwaukee, W I 53212 New York. NY 10017
Milwaukee
Frankl in
xxxx _-_ xxxx
xxxx xxxx xxxx
_-- Bergen xxxx
Cuyahoga
F a i r f i e l d
Milwaukee
Rhone-PoulencInc.Chem.Div. 201-297-0100 Monmouth Junc t ion , N J Monmouth
PO Box 125 08852 Monrmuth Junc t ion , N J 08852
Merck 6 C o . , Inc , 201-574-4000 Danv i l l e , PA 17821 Montour Danvi l le . PA 17821
Merck h Co., Inc 201-574-4000 Elkton, VA 22827 Rockingham Elkton. VA 22827
Hoffmann-La Roche I n c . 201-235-5000 Nutley, N J 07110 Essex 340 Kingsland S t .
Nutley. N J 07110
_ _ _ xxxx
_ _ _ xxxx
_ _ _ xxxx
xxxx
xxxx
xxxx
xxxx
****
_ _ _
****
42
Table 8 . (Continued)
OTHER BIOERODUCT MAKERS TELEPRONEI MANUFACTURING LOCATION COUNTY INDUSTRIAL MILITARY ==BLAST CCMPANY HEADQUARTERS TELM ATTACK ATTACK +++=FALLOUT
==HIT ==HIT ****=BOTH
---=Clear ---=Clear ---=Clear
Xanthan and o t h e r uum producers
American Cyanamid Co. 201-831-2000 Wayne, N J 07470
1ndust.Chem.Prod.
Wayne, NJ 07470
Pharmacia, Inc .
BOO-T Centenn ia l Ave.
Piscataway. N J 08854
Hercules Inc.
Hercules Plaza Wilmington, DE 19894
- - - **** 201-457-8000 Piscataway, NJ 08854 Somerset XXXX
--- **** 302-594-6500 Wihing ton , DE 19894 New C a s t l e X X X X
Merck & Co. , Inc . , 619-292-4900 San Diego, CA
Kelco Div.. 8355-T Aero D r . San Diego, CA 92123
San Diego X X X X - - _ ****
Merck 6 C o . , I n c . Kelco Div.,
Okmulgee, OK 74447
--- - - - 619-292-4900 Qkmlgee, OK 74447 Okmulgee XXXX
Rhone-Poulenc Inc . 201-297-0100 Monmouth Junc t ion , N J Monmouth X X X X - -_ **** Chem. Div . , POB 125 08852
H o m u t h J u s c t i o n . N J 08852
4 3
to expedient antibiotic production must have a sterilizable fermenta-
tion section in place. The specific fermentation equipment required
for the production of the different antibiotics is similar. The
equipment in an aerobic fermentation unit should be able to produce
any of the desired antibiotics. The requirements are (1) the organism
to produce the antibiotic, (2) sterile conditions for culture trans-
fers, ( 3 ) ability to sterilize all equipment, ( 4 ) sterile air supply,
(5) fermenter agitation and cooling and chilled water capacity (with
either internal coils or vessel jacket), and (6) basic instrumentation
for process control (oxygen tension, pH, temperature, pressure, etc.) . The separation and purification section of an antibiotic plant
is usually large (of comparable size and cost to the fermentation
section), contains many steps, and is specific to the antibiotic
being produced. In addition, the degree of purity required varies
with the application and delivery form of the antibiotic. The major
retrofit required to convert an existing fermentation plant to anti-
biotic production is the addition of a separation and purification
section. For the conversion to be feasible under the constraints of
the time limits involved, a substantial reduction in size and com-
plexity of the separation and purification unit must be achieved.
The most likely way to accomplish this is to make substantial reduc-
tions in the purity of the final antibiotic product. Reduced-purity
products can only be tolerated in antibiotics which are taken orally.
Impure products can be made by spray-drying the filtered broth,
thereby greatly simplifying the production procedure. Spray-drying
is commonly used in the enzyme, SCP, and veterinary antibiotic
industries, and it is a readily available technology requiring only
a large source of clean, hot, dry gas and a large cylindrical vertical
chamber into which the material can be sprayed in the form of small
droplets. Spray-dried antibiotics could only be given as oral anti-
biotics because of the lack of complete purity and some adverse
reactions could be expected. If existing antibiotic-producer plants
are intact or easily repaired, however, injection quality antibiotics
could be produced at these locations.
44
Other possibilities for reducing the size and complexity of
expedient separation and purification equipment involve employing
either HPLC or supercritical fluid (SFC) extraction to perform the
separation and purification from the filtered or unfiltered fermen-
tation broth.35-43 These new separation methods could substantially
reduce the size and complexity of an antibiotic plant without sacri-
ficing product quality. However, both HPLC and SCF extraction are
presently in the developmental stage for antibiotic separations.
6 . 3 SINGLE-CELL PROTEINS
Facilities that produce single-cell protein are probably the
best candidates for conversion to anitbiotic production following a
nuclear disaster. These plants have fermentation facilities which
operate under highly aerobic and sterile conditions to grow microor-
ganisms which are similar to those used to produce antibiotics.
Both the antibiotic and SCP fermentation processes deal solely with
pure cultures, while most other fermentation industries deal with
the much simpler mixed culture fermentations. The sterilization
equipment is in place for sterilizing the air, media, nutrients, and
the downstream separation processing equipment. The production of
antibiotics is highly aerobic requiring large quantities of compressed
sterile air, and SCP plants have this capacity. These plants also
have all of the necessary laboratory facilities required for chemical,
biochemical, microbiological, and quality assurance analyses required
during the production of sterile products. The equipment necessary
to operate at the subambient conditions of antibiotics plants ( 0 -
25°C) is also present. Some separation equipment is also available,
especially filters and centrifuges for separating cells from the
media, spray- drying facilities to produce a dry stable product, and
often ultrafiltration units for producing very pure macromolecular
products (like antibiotics).
The SCP plant located at Hutchinson, Minnesota, has been iden-
tified as a top candidate for conversion to antibiotic production
after a national disaster. This plant is outside any of the nuclear
attack zones and contains two 75,700-liter (20,000-gallon) state-of-
45
the-art fermenters. The surrounding area can provide most of the
raw materials required, and a constant source of 1 0 ° C cooling water
is present. The cooling water source is quite important because of
the large chilled water requirements in antibiotic plants. If
chilled water has to be produced by refrigeration, it represents a
large energy requirement. Detailed information on the Hutchinson
SCP plant is given in Appendix H.
47
7 . ALTERNATE SOURCES OF ANTIBIOTICS
Under conditions when it becomes impossible to produce enough
antibiotics in the postattack environment, two additional alternatives
exist. There is substantial antibiotic-manufacturing capacity in
Mexico and in Puerto Rico. Table 9 lists the headquarters and
telephone numbers of the four antibiotic manufacturers in Puerto
Rico and the Mexican manufacturers. Consideration should be given
to importing antibiotics from these locations after a nuclear attack.
Small planes can carry significant quantities of dry crystalline
antibiotics.
Antibiotics can be stored for many years in bulk crystalline
form at low temperatures. In planning for a nuclear attack, con-
sideration should also be given to building stockpiles of antibiotics
stored in a manner which will ensure a shelf life of many years. In
any case several low volume antibiotics which have unique properties
should be stockpiled. For example, lincomycin, tobramycin, and
spectinomycin are used for the treatment of some severe infections
in penicillin allergic patients.
48
Table 9 . Alternate antibiotic production sites outside of the
continental U.S
Antibiotic producer Telephone/ Manufacturing Antibiotics
company headquarters telex location produced
F'uerto Riw Abbott Laboratories ( 8 0 0 ) 323-9065 Barceloneta Erythromycin and
1400 Sheridan Road ( 9 1 0 ) 235-1584 00617
North Chicago, IL 60064
its salts
Eli Lilly and Company ( 3 1 7 ) 261-2000 Carolina 00630 Tobramycin sulfate
307 E. McCarty Mayaguez 00708 Erythromycin
Indianapolis, IN 46206 estolate,
Cephalexin
The Upjohn Company ( 6 1 6 ) 323-4000 Arecibo 00612 Clindamycin,
7000 Portage Road Lincomycin
Kalamazoo. MI 49001
E. R. Squibb h Sons ( 6 0 9 ) 921-4000 Humacao 00661 Nystatin,
P.O. Box 4000 Amphotericin b
Princeton, NJ 08540
Mexico
Orsabe. S. A.
Beneficiadora e
Industrializadora S . A .
de C.V.
Fermentaciones y
Syntasis, S . A .
Femic S . A . de C.V.
Quinonas de Mexico
Cuernavaca. Mexico Synthetic Penicillins
Carretera Mexico- Gentamicins
Laredo, Mexico
Mexico Cephalosporin C
Synthetic Penicillins
Ixapalapa. Mexico Cephalosporin C, EES. Synthetic Penicillins
Tetracyclines
Mexico City, Mexico Synthetic Penicillins
49
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2.
3.
4.
5 .
6.
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1.
2 .
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55
8 .3 PENICILLINS
1.
2 .
3 .
4 .
5 .
6 .
7 .
8 .
9 .
10.
11.
1 2 .
A. M. Cantwell, R. Calderone, and M. Sienko, "Process Scale-up of a P-Lactam Antibiotic Purification by High-pressure Liquid Chromatography," Journal of Chromatographv, 316 1 3 3 ( 1 9 8 4 ) .
N. R. Chatterjee, "A Simple Method for Purification of Sodium Penicillin from Penicillinic Acid Contaminant," Hindustan Antibiotics Bull. , 14 ( 3 ) 1 4 3 ( 1 9 7 2 ) .
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J. C. Dwyer, C. Findeisen and W. Skea, "Chromatographic Purifica- tion of BetaLactam Antibiotics," Pharmaceutical Technology. 6 ( 9 ) 100 ( 1 9 8 4 ) .
R. P. Elander, "Strain Improvement and Preservation of 8-Lactam- Producing Microorganisms," Handbook of Experimental PharmacoloEv. - 67 97 ( 1 9 8 3 ) .
A. L. Elder, "The History of Penicillin Production," Chemical Engineering Progress - Svmposium Series, 66 (100) 1 ( 1 9 7 0 ) .
H. M. El-Saled, M. K. El-Marsafy, and M. M. Darwish, "Chemically Defined Medium for Penicillin Purification," Die Starke 3 ( 8 ) 282 ( 1 9 7 6 ) .
A. R. Giona, R. DeSantis, L. Marrelli and L. Toro, "The Influence of Oxygen Concentration and of Specific Rate of Growth on the Kinetics of Penicillin Production," BiotechnoloEv and Bioengineering, 18 4 9 3 ( 1 9 7 6 ) .
A. R. Giona, L. Marrelli, and L. Toro, "Kinetic Analysis of Penicillin Production by Semicontinuous Fermenters," Biotech- nology and Bioenvineering, 18, 4 7 3 ( 1 9 7 6 ) .
S . Hasegawa, K. Shimizu, T. Kobayashi and M. Matsubara, "Efficiency of Repeated Batch Penicillin Fermentation Using Two Fermenters, Computer Simulation and Optimization," J. Chem. Tech. Biotechnol. 35B 33 ( 1 9 8 5 ) .
E. Hegewald, B. Wolleschensky, R. Guthke, M. Neubert, and W. A Knorre, "Instabilities of Product Formation in a Fed-Batch Culture of Penicillin Chrysogenum," Biotechnology and Bioengi- neerinE 23 1 5 6 3 (1981) .
G. J. M. Hersbach, C . P. van der Beek, and P. W. M. van Dijck, "The Penicillins: Properties, Biosynthesis, and Fermentation," pp 4 5 - 1 4 0 in Biotechnology - of Industrial Antibiotics ed E. J. Vandamme, Marcel Dekker, Inc., New York, 1 9 8 6
56
13
14
15
1 6
17
18
19.
20.
21.
22.
23.
24.
25.
J . J. Heijnen, J . A . Roels, and A . H. Stouthamer, "Application of Balancing Methods in Modeling the Penicillin Fermentation," Biotechnology and Bioenpineering 21, 2175 (1979).
J . H. Oldfield, "The Penicillin Fermentation," Science and Australian Technology. 14 (4) 8 (1977).
S . J . Pirt, "The Penicillin Fermentation: A Model for the Development of Antibiotic Fermentations," Kem. Ind.. 34 (1) 13 (1985).
S . J. Pirt,and B. Mancini, "Inhibition o f Penicillin Production by Carbon Dioxide," Journal o f Applied Chemical Biotechnology - 25 781 (1975).
M. A . Qadeen, A . Ghafoor, and M. 1. D. Chughtai, "Use o f Penicillin Waste Mycelium in Fermentation Media: Production of Chlortetracycline by Streptomyces Aureofaciens," Pakistan Journal o f Biochemistrv, 3 (1-2) 42 (1970).
T. A. Savidge, "Enzymatic Conversions Used in the Production o f Penicillins and Cephalosporins," Drugs and the Pharmaceutical Sciences. 22 171 (1984).
J . C. Sheehan and K. B. Henery-Logan, "The Total and Partial General Synthesis of the Penicillins," Journal of the American Chemical Society, 84 2983 (1962).
M. D. Smith and C. S . Ho, "On Dissolved Carbon Dioxide in Penicillin Fermentations," Chemical Engineerinn - Communications, - 37 21 (1985).
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PATENTS
V. Ardelean, A. Georgeta, F. Dogaru, M. Onu and G . 1. Agape, "Culture Medium for Penicillin G Production" British Patent # 1276397 July 13, 1970.
C. L. Dunn, M. Souders, and C. Groot, "Methods of Concentration," US Patent #2,481,092 September 6, 1949.
G. J. Pierotti, M. Souders, and F. A. French, "Purification of Penicillin," US Patent #2,488,558 November 22, 1949.
G . J. Pierotti, "Extraction o f Penicillin," US Patent #2,503,214 April 4, 1950.
57
2 6 .
2 7 .
2 8 .
2 9 .
8.4
1.
2 .
3 .
4 .
5 .
6
7
8 .
G. J. Pierotti, E. A. Anderson, and M. Souders, "Purification of Penicillin," US Patent # 2 , 5 0 3 , 2 1 5 April 4 , 1 9 5 0 .
G. J. Pierotti, R. A. Wilson, and E. A. Anderson, "Purification of Penicillin," U.S. Patent # 2 , 5 0 3 , 2 1 6 April 4 , 1 9 5 0 .
M. Souders, "Penicillin Extraction Process," US Patent #2 4 8 8 559 November 2 2 , 1 9 4 9 .
M. M. Young, "Fermentation Processes for Penicillin and Cel- lulose, US Patent #3 975 2 3 6 January 2 6 , 1 9 7 6 .
CEPHALOSPORIN
A. L. Demain, "Biochemistry of Penicillin and Cephalosporin Fermentations, Llovdia 37 ( 2 ) 147 ( 1 9 7 4 ) .
M. Kalyanpur, W. Skea and M. Siwak, "Isolation of Cephalosporin C from Fermentations Broths Using Membrane Systems and High- Performance Liquid Chromatography," DeveloDments in Industrial Microbiologv, 26 4 5 5 ( 1 9 8 5 ) .
T. Kanzaki, and Y. Fujisawa, "Recent Progress in Cephalosporin Fermentations,"J. Takeda. Res. Lab. 34 (2) 3 2 4 ( 1 9 7 5 ) .
K. Kitano, K. Kintaka, S . Suzuki, K. Katamoto, K. Nara, and Y. Nakao, "Production of Cephalosporin Antibiotics from N-Paraffins by Strains Belonging to the Genus Paecilomyces," J. Ferment. Technol., 52 (10) 7 8 5 ( 1 9 7 4 ) .
M. Masazumi, T. Imanaka, T. Yoshida, and H. Taguchi, "Modeling of Cephalosporin C Production and Its Application to Fed-Batch Culture," J. Ferment. Technol., 3 ( 2 ) 1 1 5 ( 1 9 8 1 ) .
M. Matsumura, T. Imanaka, T. Yoshida and H. Taguchi, "Effect of Glucose and Methionine Consumption Rates on Cephalosporin C Production by Cephalosporin Acremonium," J. Ferment. Technol., - 56 ( 4 ) 345 ( 1 9 7 8 ) .
R. 0 . Miller and N. Neuss, "High Performance Liquid Chromatog- raphy of Natural Products. I. Separation of Cephalosporin C derivatives and Cephalosporin C form Antibiotics; Isolation of Cephalosporin C From Fermentation Broth," Journal o f Antibiotics, - 29 ( 9 ) 9 0 2 ( 1 9 7 6 ) .
D. A . Preston and W. E. Wick, "Laboratory Evaluation of Cephalosporin Antibiotics," Developments in Industrial Microbiologv, Volume 16, American Institute of Biological Sciences, Washington, D.C. ( 1 9 7 5 ) .
58
9 .
10.
11.
1 2 .
1 3 .
14.
1 5 .
8.5
1.
2 .
3 .
4 .
5 .
T. A. Savidge, "Enzymatic Conversions Used in the Production of Penicillins and Cephalosporins," Drugs and the Pharmaceutical Sciences, 22 171 ( 1 9 8 4 ) .
A . Trilli, V. Michelini, V. Mantovani and S . J. Pirt, "Estimation of Productivities in Repeated Fed Batch Cephalosporin Fermentation," J. APDI. Chem. Biotechnol., 27 219 ( 1 9 7 7 ) .
M. E . Wildfeuer, "Approaches to Cephalosporin C Purification from Fermentation Broth," Purification of Fermentation Products, 156 ( 1 9 8 5 ) .
R. B. Woodward, K. Heusler, J. Gosteli, P. Naegezi, W. Oppolzer, R. Ramage, S. Ranganathan and H. Vorbruggen, "The Total Synthesis of Cephalosporin C," Journal of the American Chemical Society, - 88 ( 4 ) 852 ( 1 9 6 6 ) .
PATENTS
D. W. Blackburn, "Cephalosporin Purification Process," US Patent #4 028 355 July 18 , 1975 .
H. F. Niss, "Process for the Manufacture of Cephalosporin C," US Patent # 3 539 694 June 2 3 , 1967 .
G. T. Stewart, "Purified Cephalosporins and Their Production," British Patent #1 232 656 May 2 3 , 1967 .
TETRACYCLINES
M. Bosnjak, "Repeated Fed Batch Culture as a Possibility for High Yield Tetracycline Fermentations," Prehrambeno-Tehnoloska Reviia, 22 ( 3 - 4 ) 117 ( 1 9 8 4 ) .
D. J. D. Hockenhull, The Fermentation of the Tetracyclines. Interscience Publishers Inc., New York ( 1 9 5 9 ) .
J. H. Knox and J. Jurand, "Separation of Tetracyclines by High- Speed Liquid Chromatography, Journal of Chromatoprauhy, 110 103 (1975).
A . Kremen, "Thermodynamic Model of the Production of the Tetracycline Type," Journal of Theoretical Biology, _. - 31 363 ( 1 9 7 1 ) .
S . Ochab, "The Application of Electrophoresis for Separation of Tetracycline and Polyene Antibiotics," Dissert. Pharm. Pharmacol., 23 ( 6 ) 205 ( 1 9 7 1 ) .
59
6 .
7 .
8 .
9 .
10 .
11.
1 2 .
1 3 .
14 .
1 5 .
1 6 .
8 . 6
1.
2 .
H. H. Wasserman and T. J. Lu, "On the Total Synthesis of Tetracyclines," Journal of the American Chemical Society, 108 4 2 3 7 ( 1 9 8 6 ) .
G. J. Willekens, "Separation and Quantitative Determination of Impurities in Tetracycline," Journal o f Pharmaceutical Sciences, - 6 4 (10) 1 6 8 1 ( 1 9 7 5 ) .
PATENTS
M. Gavrilescu, C . Pal, S . User, S . Ionescu and N. Margineanu, "Process for the Isolation and Purification of Tetracycline," British Patent #1 368 6 6 8 , August 2 2 , 1 9 7 2 .
A . Gourevitch and J. Lein, "Production of Tetracycline and Substituted Tetracyclines," US Patent #2 7 1 2 517 March 3 , 1 9 5 4 .
A . B. Hatch, G . A. Hunt and J. Lein, "Tetracycline Production Using Cottonseed Endosperm Flour, US Patent #2 7 6 3 5 9 1 December 7 , 1 9 5 4 .
P. Minieri, H. Sokol, and M. C. Firman, "Process for the Preparation o f Tetracycline and Chlortetracycline," US Patent #2734018 September 2 8 , 1 9 5 3 .
B. A. Sobin, A. C . Finlay and J. H. Kane, "Terramycin and It's Production," US Patent #2 5 1 6 0 8 0 November 2 8 , 1 9 4 9 .
"Process for Producing Tetracycline by Fermentation," British Patent #1 286 9 2 9 June 30 , 1 9 7 1 .
"Production of the Antibiotic Tetracycline by Fermentation," British Patent #775 1 3 9 December 1 0 , 1 9 5 3 .
S . P. A. LePetit, "Production of the Antibiotic Tetracycline by Fermentation," British Patent #775 1 3 9 , May 2 2 , 1 9 5 7 .
W. J. McGhee and J. C. Melna, "Process for the Production of Tetracycline, U.S. Patent # 2 , 7 7 6 , 2 4 3 January 1, 1 9 5 7 .
BACITRACIN
R. E. Bennett, J. F. Dudley, and M. W. Shepard, "Bacitracin! Product of Biochemical Engineering," Industrial And Eneineerine -
Chemistry. 43 ( 7 ) 1 4 8 8 ( 1 9 5 1 ) .
R. J. Hickey, "Bacitracin, Its Manufacture and Uses," Progress in Industrial Microbiolonv. 1 9 3 - 1 5 0 ( 1 9 6 4 ) .
60
3.
8 . 7
1.
2 .
3.
4 .
5.
6.
8 .
9 .
10 .
11.
12.
8 . 8
H . J. Peppler and D. Perlman, Microbial Technology Volume 1, Second Edition, Academic Press, New York 1979.
SULFONAMIDES
A . Aitiba and M. Tsurooka, "Sulfa Drugs," Science, 24 195 (1954).
P. I. Astrakhantsen, "Aid to the Pharmaceutical Industry by VNIKRFl During the Patriotic [Second War] War," Farmatsiva, - 8 ( 4 ) 12 (1945).
H. Braeuniger and K. Spangenberg, "Separation of Sulfonamides by Countercurrent Distribution," Pharmazie. 2 343 (1954).
C. Lapiere, "Analytical Study of Sulfonamides, J. Pharm. u., 2 135 (1947).
W. H. Longnecker, "Simplified Partition Chromatographic Procedures," Analvtic Chemistry, 21(11) 1402 (1949).
W. H. Powers, Advancing Fronts in Chemistry: Chemotherapy Reinhold Publishing Co., New York (1946).
D. R. Shridhar and K. S . Narang, "Studies in Sulphonamides - A New Approach, Part 1," J. Indian Chem. Soc., 33 (5) 305 (1956).
PATENTS
N. Bennett, H. Dodd, and W. C. Sprent, "Sulfonamides," British #336 512 January 1 7 , 1930.
M. Dohrn and P. Diedrich, "Sulfonamide Derivatives," German Patent #840 241 May 19, 1952.
G. Maemoto, "Refining of Sulfonamides, , 'I Japan Patent #4077 August 21, 1953.
J. Matzke, "Water-Soluble Sulfonamide Preparations," U.S. Patent #180 656 January 10, 1955.
F. Scholhofer, "Sulfonamide Solutions for Chemotherapeutical Use," German Patent #831 880 February 1 8 , 1952.
SEPARATION AND mTRIFICATION
1. J. M. Begovich and W. G . Sisson, AIChE. J., 30, 705 (1984).
61
2.
3.
4.
5.
6 .
7 .
8 .
9.
10.
11.
12.
13.
14.
A. M. Cantwell, R. Calderone, and M. Sienko, "Process Scale-up of a p-Lactam Antibiotic Purification by High-Performance Liquid Chromatography," Journal o f Chromatographv. 3L6 133 (1984) .
N. R. Chatterjee, "A Simple Method for Purification of Sodium Penicillin from Penicillinic Acid Contaminant," Hindustan Antibiotics Bull., 14 (3) 143 (1972).
H. Colin, D. G . Lowy and J. Cazes, "Design and Performance of a Preparative-scale HPLC," American Laboratory 136, May (1985).
J. Dwyer, "Process Separations of Antibiotics by HPLC Process Systems," World Biotech. Report, 651 (1984).
J. Dwyer, C . Findeisen and W. Skea, "Chromatographic Purification of Beta-Lactam Antibiotics," Pharmaceutical Technolopv, 8 (9) 100 (1984).
J. Dwyer, "Chromatographic Purification of Semisynthetic Beta- Lactam Antibiotics," Annual Reports on Fermentation Processes, - 8 93, (1985).
J. Dwyer, "High Performance Chromatography as a Process T o o l , " AIChE Symposia Series, 82 (250) 120 (1987).
I. Dzierzynska, "The Application of Electrophoresis for Separation of Tetracycline and polyene antibiotics," Dissert. Pharm. Pharrnacol, 23 ( 6 ) , 205 (1971).
F. N. Inman, "Filtration and Separation in Antibiotic Manufacture," Filtration and Separation 165 May/June 1984
M. Itamihira, M. Tanifuchi and T. Kobayashi, "Removal of Organic Solvent from Antibiotics with supercritical Carbon Dioxide," J. Ferment. Technol. 65 (1) 671 (1987).
M. Kalyanpur, W. Skea, and M. Siwak, "Isolation of Cephalosporin C from Fermentation Broths Using Membrane Systems and HPLC," SA Filtration. 8 November 1984.
M. Kalyanpur, W. Skea, and M. Siwak, "Isolation of Cephalosporin C From Fermentation Broths Using Membrane Systems and High- Performance Liquid Chromatography," Developments in Industrial Microbiolow, 26 455 (1985).
J. H. Knox and J. Jurand, "Separation o f Tetracyclines by High- Speed Liquid Chromatography, Journal of Chromatopraphv, 110 103 (1975).
62
1 5 .
1 6 .
1 7 .
1 8 .
1 9 .
20 .
21 .
22 .
23 .
24.
25 .
2 6 .
2 7 .
28 .
K. A . Larson and M. L. King, "Evaluations of Supercritical Fluid Extraction in the Pharmaceutical Industry," Biotechnolow Progress, 2 ( 2 ) 7 3 ( 1 9 8 6 ) .
C. F. Mascone, "Separations are key to Biotech Scale-up," Chem. Engr. January 1 9 , 1987.
D. J. Morecombe, "High-Efficiency Preparative Scale Reversed- Phase High-Performance Liquid Chromatographic Purification of I4C-Labelled Antibiotics," J. Chromatography 389 389 ( 1 9 8 7 ) .
D. Perlman, Advances in Applied Microbiology, Volume 11, Academic Press, New York, ( 1 9 6 9 ) .
E. S . Perry and A. Weissbeager, Separation and Purification, 3rd edition, John Wiley and Sons, New York, ( 1 9 7 8 ) .
R. R. Rasmussen, and M. H. Scherr, "Preparative Low Pressure Chromatography o f Antibiotics on a Column o f Diol-Bonded Silica Gel," J. Chromatography 386 325 (1987).
C. D. Scott, R. D. Spence, and W. G . Sisson, J. ChromatoFraDhv, 126 3 8 1 ( 1 9 7 6 ) .
C. K. Shih, C. M. Snavely, T. E. Molnar, J. L. Meyers, W. B . Caldwell, and El L. Paul, "Large-Scale Liquid Chromatography System," m, 53 October ( 1 9 8 3 ) .
I. Sunshine, Handbook of Analytical Toxicolow, CRC Press, Boca Raton, FL, ( 1 9 7 9 ) .
E. Tomlinson and A . Regosz, "Antibiotics: 1 j3-Lactam Antibiotics," Solubility Data Series, Pergamon Press, New York (1985).
J. V. Uri, "Novel Fermentation Procedures for the Production and the Isolation of Antibiotics," Acta Microbiologica Hungarica, - 33 ( 4 ) 305 , ( 1 9 8 6 ) .
J. M. Walsh, G. P. Ikonomov and M. D. Donohue, "Supercritical Phase Behavior: The Entrainer Effect," Fluid Phase Equilibria - 33 295 , ( 1 9 8 7 ) .
I. P. Watson, D. J, Platt, S . J. McIntosh, M. J. Stewart, and H. N. Cohen, "High-Performance Liquid Chromatography as an Aid to the Rational U s e of Antimicrobial Agents," Anal. Proc., 180 April 1982.
M. N. Weiss, "Process Scale Liquid Chromatography," Industrial Chemist 8 ( 8 ) 1 6 ( 1 9 8 7 ) .
63
2 9 .
3 0 .
3 1 ,
3 2 .
33.
3 4 .
8 . 9
1.
2 .
3.
4 .
5.
E. R. White and J Chromatography of (7) 836 (1981).
. E. Zarembo, "Reverse Phase High Speed Liquid Antibiotics," The Journal of Antibiotics, 34
M. E. Wildfeuer, "Approaches to Cephalosporin C Purification from Fermentation Broth," Purification of Fermentation Products, 156 (1985).
G. J. Willekens, "Separations and Quantitative Determination o f Impurities in Tetracycline," Journal of Pharmaceutical Sciences, - 6 4 (10) 1681 ( 1 9 7 5 ) .
T. T. Yoshikawa, S . K. Maitra, M. C. Schotz, and L. B. Guze, "High-pressure Liquid Chromatography for Quantitation of Antimicrobial Agents," Reviews of Infectious Diseases, 2 ( 2 ) 169 (1980).
PATENTS
H. Coenen, "Isolation and Purification of Antibiotics," German Patent #33 18 194 May 19, 1983.
S. Pines, "Antibiotic Purification Process," US Patent #3 9 8 3 108 September 28, 1976.
PRESERVATION
W. D. Bellamy, "Preservation of Foods and Drugs by Ionizing Radiations," Advances in Amlied Microbiology, - 1. 4 9 ( 1 9 5 9 ) .
B. A. Dinel, D. L. Ayotte, R. J. Behme, B. L. Black, and J. L. Whitby, D r w Intelligence Clinical Pharmacy, 11 ( 9 ) 5 4 2 ( 1 9 7 7 )
D. J. D. Hockenhull, "Production of Antibiotics by Fermentation," Essays in Amlied Microbiologv, 411 John Wiley and Sons, New York ( 1 9 8 1 ) .
C. J. Holmes, R. K. Ausman, C. W. Walter, and R. B. Kundsin, "Activity of Antibiotic Admixtures Subjected to Different Freeze-Thaw Treatments," Drug Intelligence and Clinical Pharmacy, - 14 (5) 353 (1980).
P. W. Muggleton, "The Preservation of Cultures," Progress - in Industrial Microbioloev, 191 ( 1 9 6 2 ) .
8.10 STERILIZATION
1. G. B. Cherry and S. D. Kemp, "The Sterilization of Air," Progress -
in Industrial Microbiologv, ir. 37 ( 1 9 6 3 ) .
64
M. Herold and J. Necasek, "Protected Fermentation," Advances in Apulied Microbiology, - 1 1 (1959).
G . L . Solomons, "Fermentation Equipment," Advances in Applied Microbiology, 14 231 (1971).
G. R. Wilkinson and L. C. Baker, "Modern Trends in Steam Sterilization," Progress - in Industrial Microbiolony 5 231 (1964).
8.11 ANALYTICAL METHODS
H. P. Fiedler and A. Plaga, "Separation of Amino Acids and Antibiotics by Narrow-Bore and Normal-Bore High Performance Liquid Chromatography with Pre Column Derivatization," J. Chrorn. 386 229 (1987).
P. W . Later, B. E. Richter, D. E. Knowles and M. R. Anderson, "Analysis of Various Classes of Drugs by Capillary Supercritical Chromatography," J. Chrom. Sci. 24 249 (1986).
H. Oka, Y. Ikai, N. Kawamura, K. Uno and M. Yamada, "Improvement of Chemical Analysis of Antibiotics: Determination of Eight Tetracyclines using Thin-Layer and High-Performance Liquid Chromatography," J. Chrom. 393 285 (1987).
P . A . Ristuccia, "Liquid Chromatographic Assays of Antimicrobial Agents," J. Liquid Chrorn., 10 (2 and 3) 241 (1987).
R. R. Ryall and D. M. Radzik, "Critical Evaluation of LC with W - V I S Photodiode-Array Detection in Pharmaceutical Analysis," Chromatography, April, 28 (1987).
D. Sampson and R. Benn, "HPLC of Antibiotics," HiF.h Perform. L i r l . Chromatography Clin. Lab. 82 (1986).
R. E. Schirmer and A. G. Gargus, "Applications o f Remote Sensing Using Fiber Optics and W-VIS-NIR Spectroscopy," American Laboratory December 30 (1986).
8. I. Sunshine, Methodology for Analytical Toxicology Volumes I and 111, CRC Press, Boca Raton, FL, (1975).
9. I . Sunshine, Handbook o f Analytical Toxicology, CRC Press, Boca Raton, FL (1979).
APPENDIX A
Production of Penicillin Antibiotics
67
Appendix A. PRODUCTION OF PENICILLIN ANTIBIOTICS
The production of penicillin begins from a master stock of the
production organism from which vegetative mycelium is prepared in a
series of inoculum development steps to increase the concentration of
fungal mycelium. The inoculum development is carried up through shake
flasks and then into an agitated vessel with a medium containing
sufficient fermentable carbohydrate, an organic nitrogen source,
calcium carbonate as a buffer to prevent low pH values, and various
inorganic salts as in Tables A - 1 and A - 2 . The presence of about 1%
carbon dioxide appears essential for germination of spores, and the
carbon dioxide must be kept below 4% to prevent strong inhibition
during penicillin production. A critical spore concentration (=5 x
l o 9 spores/m3 ) occurs above which more filamentous mycelium are
formed, enzyme levels are high and penicillin production is good in
the production reactor. Below this concentration, dense pellets are
formed, glucose utilization is less efficient and enzyme levels and
penicillin production are lower. The spore concentration is only
important in the first shake flask inoculation. An inoculum volume
of 5 to 10% of the production fermenter (at about 20 kg mycelium dry
weight/m3 product from the inoculum reactor) is required to give a
high specific penicillin production rate in the production fermenta-
tion. It is very important that the culture still be i n the unin-
hibited growth phase ( l o g growth) when the transfer is done.
The present fermentation process is a fed-batch process o r a
repeated fed-batch process (a portion of the broth is withdrawn at
intervals). A fermenter of 1 5 0 to 200 m3 is used at about 80% full
( 1 2 0 to 160 m3 of broth) with 3- to 4-kW/m3 power input. Oxygen is
supplied by air flow at 30 to 60 m3/m3 broth per hour (0.5 to 1.0 VVM)
at 300 kPa ( 4 3 psia) pressure with a fermenter pressure of 135 to 170
kPa ( 1 9 . 5 to 2 4 . 5 psia) and an oxygen uptake rate of 25 to 50 m o l / m 3
broth per hour. The initial mycelium concentration in the fermenter
is 1 to 2 kg dry weight/m3. The other conditions include broth tem-
perature of =25"C, pH of 6 . 5 , and a continuous feed glucose concen-
tration of 500 kg/m3 solution with a glucose feed rate of 1.0 to 2.5
68
Table A-1. Raw materials needed for penicillin production
(120-160 m3 batch-yielding - 28 kg Pen G Na salt/m3 - 3100-4200 kg) (-20,000-25.000 units per ml)
~~
Material @antity (approximate) Used for
Gluco s e 2.5kg/m3 broth per hour(200 hr -56,000 kg/batch
Fermentation
Phenylacetic acid Acetic and/or Lactic acid Amino acids, peptides,amines Amnon i a Corn-steep liquor solids Antifoam-consumable oil Variable by continuous feed Minerals for defined medium Filter-aid A m y l or butyl acetate
Activated charcoal Potassium or sodium acetate Isopropyl or butyl alcohol Sulfuric or phosphoric acid
-0-6kg/m3 - 500 kglbatch
40-50kg/m3 - 5000 kg/batch
3 0 - 4 0 m3 ( 3 0 - 4 0 . 0 0 0 L/8-10,000 gal)
Fermentation Fermentation Fermentation Fermentation Fermentation Fe m e n ta t ion Fermentation Filtration Solvent - extraction Purification Crystallization Crystallization pH adjustment
Table A-2. Alternate materials for defined medium for penicillin production
Component Concentration (kg/m3) Batch requirement (kg)
Na2S04 0 - 1 up to 160 kg (NH4 )2SO4 0-10 up to 1600 kg NH4N03 or KN03 0-8 up to 1280 kg ~ 2 P O 4 . NaH2m4, K ~ m 4 0 . 4 - 8 up to 1280 kg CaC03 0-5 up to 800 kg @so4 7n20 0.25-0.8 up to 128 kg FeS04"7H20 /Fe(NH4)2(S04)2 0 . 0 4 - 0 . 4 up to 64 kg ZnS04'7H20 0.01-0.05 up to 8 kg CuS04 5H20 0.005-0.01 up to 1300 g MnS04' 4H20 0.01-0.06 up to 9600 g corn4 7n20 0-0.005 up to 800 g CaC12 0-0.05 up to 8000 g
NOTES: Alternate carbon sources--Lactose, Ethanol, Acetic acid or acetate, Pentoses, Hexoses, disaccharides (maltose, etc.). plyols. dextrins, starch hydrolysates, and molasses. (Ethanol or fatty oils cause a higher oxygen demand and heat generation than glucose or other sugars.)
Alternate nitrogen sources--Cottonseed meal, brans with phytic acid and phosphorus, Pharmamedia,Penicillim mycelium, and ammonia, amnonium sulfate.
Precursor Sources--1-phenyl-n-decane and 1-phenyl-n-dodecane.
69
kg/m3 broth per hour (1.8 kg/m3 per hour optimum) and a total fer-
mentation duration of 180 to 220 hours. Tables A - 1 and A - 2 give the
approximate requirements for the chemicals needed for the fermenta-
tion. Figure A - 1 shows the overall process.23
Productivity in the penicillin fermentation depends upon the
genetic capability of the culture, biomass concentration, limited
mycelial growth rate, and mycelial mass during the rapid growth phase
ending at the optimum mass limit of about 80% of the maximum. Also,
starting at the optimum glucose concentration for the fermentation,
maintaining the optimum glucose and nutrient feed rate during the
fermentation, and having the appropriate precursor affect produc-
tivity. Specific oxygen uptake rates and carbon dioxide evolution
rate are also associated with productivity. Temperature control is
important for obtaining the maximum production rate of penicillin,
and the metabolic heat and the mechanical heat generated by agitation
and aeration must be removed from the reactor. However, this becomes
more difficult as the viscosity of the mycelial mass increases dur-
ing the fermentation.2’5*e’35,44
Several control algorithms for the carbon feed have been devel-
oped, based on dissolved oxygen, pH, carbon dioxide evolution, the
growth curve, and the specific growth rate. The uptake rates for
the other nutrients besides carbon (glucose) are shown in Table A-3.
Shown in Fig. A-2 in a typical concentration profile of the fed-
batch penicillin fermentation with some of the control parameter
profiles shown. Tables A - 4 and A - 5 give some of the common yields
for penicillin G and V from glucose, and the distribution of glucose
into penicillin, mycelium, and maintenance energy during a
fermentation.
Product Recovery Processes
The recovery of penicillin from a fermentation broth takes
about 15 hours. Gist-Brocades‘ state-of-the art process is described
below, and shown in Fig. A - 3 . The first step of the separation is
the filtration of the mycelium from the broth on a rotary vacuum
filter using filter aid or precoats and on which the mycelium is
70
ORNL D W G 8 7 - 4 2 0
FEED-SUBSTRATES, ETC.
SEED VESSEL
LABORATORY CULTURE. SPORES T O INOCULUM
PRODUCTION FERMENTOR
ACTIVATED CLARIFIER CARBON
ROTARY VACUUM FILTER SOLVENT
EX TRACTION
CARBON TREATMENT
1 I
-- T O CHEMICAL MODIFICATION DRYER
I
W CRYSTALLIZER 'i BLENDER
I
OUALITY ASSURANCE TESTING
BULK STERILE P R 0 DUCT
Fig. A-1. General production scheme for penicillin production.
71
Table A-3. Specific uptake rates for nutrients during a penicillin fermentationa
Nutrients and precursor
~ ~~
Specific u take rates
mycelium dry weight/h)
(at p=O.O15 hr E ; in mmol/g
Carbon (sugar)
Oxygen
Nitrogen (as NH,)
Phosphorus (as PO, - )
Sulfur (as
Precursor (Phenylacetic acid)
~~
0.33
1. 60b
0 . 1 2
0.006
0.029
0.013
a D. D. Y. R y u and J. Hospodka, "Quantitative physiology of Penicillium chrvsoPenum in penicillin fermentation," Biotechnol. BioenE. 22 289-298 (1980).
For maintenance, growth and penicillin production.
72
ORNL DWG 88-288
W
0 cn U C cu cn I- z 3 t U
U I-
U
a
-
a
m a
-
100
80
60
40
20
I I I 1 I I I I I I I I I I I I d - - .e*-
0 ./. DISSOLVED OXYGEN (% saturation) 0 /. -
PENICILLIN (g/L x 1 0 ) ./' -/.' / ' /.
/. /.
0 20 40 60 80 100 120 140 160
FERMENTATION TIME (h) Fig. A-2. Typical concentration profile of fed-batch penicillin
fermentation. Source: D. D. Y. Ryu and J. Hospodka, "Quantitative physiology of Penicillium chrvsoqenum in penicillin fermentation, " Biotechnol. Bioeng. 22 289-298 ( 1 9 8 0 ) .
7 3
Table A-4. Carbon distribution into penicillin, mycelium, and maintenance energy.a
Percentage of carbon fed that is consumed for Penicillin Mycelium Maintenance Remarks
6 2 5 - 2 9 6 5 - 6 9 140 hr fermentation
10-11 2 0 - 2 8 6 1 - 7 0 200 hr fermentation aminoadipic acid recycled
16 27 5 7 aminoadipic acid discarded
aD. D. Y. Ryu and J. Hospodka, "Quantitative physiology of Penicillium chrvsogenum in penicillin fermentation," Biotechnol. Bioeng. 22 2 8 9 - 2 9 8 ( 1 9 8 0 ) .
Table A-5. Maximum and real yields of penicillin from glucosea
Method Units pen G or V Mole pen G or V g pen G-Na (per mg glucose) (per mole glucose) ( u er g g - lucose)
Maximum from 7 0 0 - 2 1 9 8 0 . 2 0 7 - 0 . 6 6 7 0 . 4 1 0 - 1 . 3 2 model calculation
Model calculation 9 5 - 2 0 0 0 . 0 2 9 - 0 . 0 6 1 0 . 0 5 7 - 0 . 1 2
Experimental 8 0 - 2 1 2 0 . 0 2 4 - 0 . 0 6 4 0 . 0 4 8 - 0 . 1 1 5
aG. J. M. Hersbach, C . P. van der Beek, and P. W. M. van Dijck, "The Penicillins: Properties, Biosynthesis, and Fermentation," pp 45 - 1 4 9 in Bictechnclonv cf Industrial AntibiotLcs ed E. J . Vandaxr,e, Marcel Dekker, Inc., New York, 1 9 8 6 .
7 4
ORNL D W G 8 8 - 2 3 7
SALT
I t ACID
TO SOLVENT RECOVERY
DISTILLATION
J 00 c x 3
TO SOLVENT RECOVERY
7 5
also washed. Penicillin is extracted in the acid form from the fil-
trate under very carefully controlled conditions of temperature, pH,
and sterility to minimize chemical and enzymatic degradation of the
penicillin in the pumps, vessels, and the extractor. The extractant
is amyl acetate in a continuous countercurrent multistage centrifugal
extractor at pH 2.5 to 3.0 and at 0 to 3°C. Centrifugal contactors
such as the Podbielniak extractor with throughputs up to 9 8 , 5 4 0 L/h
(26,000 gal/h) are used. Partition of penicillin between organic
and water phases is shown in Figures A-4 and A - 5 . Demulsifying
agents are used to prevent emulsion formation and to obtain a high
separation efficiency, and dilute sulfuric or phosphoric acid are
used to control the pH. The water phase and the organic are fed to
the extractor at a 4:l ratio, using short residence times to prevent
degradation of the penicillin under the acid conditions of the
extraction. It is also possible to extract the whole broth from the
fermenter with the solvent without going through a filtration step.
A problem of this method is that the components of mycelium will
also be extracted and will contribute impurities that must be removed
during the purification steps.
Following extraction, the penicillin-containing solvent is
treated with a slurry of activated charcoal, filtered on a pre-
coated rotary vacuum filter, and washed with solvent. Potassium or
sodium acetate is added to the solvent, and penicillin G or V is
crystallized as the salt from the solvent phase, with critical
parameters including potassium or sodium concentration, pH value,
penicillin concentration, and temperature. The crystals are separated
on a rotary vacuum filter. The crystals are washed and predried with
anhydrous isopropyl alcohol, butyl alcohol, or other volatile solvent
and then treated with fresh solvent on a horizontal vacuum belt fil-
ter and dried with warm air, resulting in a 9 9 . 5 % pure penicillin.
Spent solvents are processed for solvent purification by washing,
distillation, and drying. Table A - 6 describes the steps in the tra-
ditional purification process and Fig. A - 6 shows the stages in a
typical extraction. 0
50
20
10
5
1
0.5
0.1
0.05
7 6
ORNL DWG 88-280
1 I I I I I 1
I I I I I I 2.0 2.6 3.2 3.8 4.4 5.0 5.6 6.0
PH Fig. A-4. Distribution coefficients vs. pH of penicillin G
betxeen water and solvents. Source: R. L. Feder , IZec~-;er-; of Penicillin--Diostribution Coefficients and VaDor-Liauid Eauilibria. M.S. Thesis, Polytechnic Institute of Brooklyn, (June, 1947).
77
100
10.0
1 .o
0.1 1 2 3 4 5 6 7 8
PH Fig. A-5. Partition of penicillins as a function of the pH of
the extraction solvent (isoamyl acetate). Source: M. Souders, G. J. Pierotti, and C. L. Dunn, “The Recovery of penicillin by extraction with a pH gradient,’ pp 3 9 - 4 2 in The history of uenicillin uroduction, Chemical Engineering Progress Symposium Series, V o l . 6 6 , No. 100, American Institute of Chemical Engineers, New York, 1970.
78
Table A-6. Traditional penicillin purification
~~~
Step Purpose Equipnent Basis Size
Filtration Separation of mycelia Continuous, rotary
from penicillin- vacuum d r m filter
containing broth
to 148 m2
(1600 ft')
Extraction Separation of Continuous. multistage Differential extraction: t o m m penicillin from other countercurrent when pE>pKa. ( 2 6 0 O O g a l ~ )
soluble components extractors penicillin more soluble
in organic phase: when
pFI,pKa, penicillin
m r e soluble in
aqueous phase
Crystallization Further purification Tanks and and stabilization gravitational
separators
Via addition of sodium
or potassium ions
Drying Stabilization Vacuum o r w m - a i r Anhydrous solvent
driers
79
ADDITIVES H,SO,
FRESH SOLVENT
9 ?
ORNL D W C HH ? H I
LEAN BUFFER
? ? * FILTERED
w 1 STAGE 2 STAGE 1
PENICILLIN BROTH
TO SOLVENT RECOVERY STAGE 3
-I
71
4 ?
J-- SOLVENT
SPENT BUFFER
Fig. A-6. Traditional three-stage extraction of penicillin. Source: S. Queener and R. Swartz, "Penicillins: Biosynthetic and semisynthetic," pp 35-122 in Economic microbiology, V o l . 3. ed A. H. Rose. Academic, New York, 1979,
Three types of indirect assay methods have been used for peni-
cillin quality control. They the iodometric techniques, mercury
imidazole and the ferric hydroxamate methods. These have been
improved through the years so that automatic analyses can be done.
Direct ultraviolet absorption is used to detect penicillins as they
are separated on reverse-phase HPLC columns containing octyl and
octadecyl bonded material as the stationary phase. HPLC is able to
separate the side-chain precursor, penicillin, and penicillin by-
products or degradation products in one analysis. Because of this,
it is a fast, easy to learn, reliable method for quality assurance.
Immobilized penicillinase can also be used in combination with a pH
electrode or a thermistor to measure the concentration of the
penicillin in the fermenter or fermentation broth.
Modification of Penicillin G to Produce Semisynthetic Penicillins and
Cephalosporins.
The semisynthetic penicillins are produced with 6-aminopenici-
lanic acid (6-APA) as the starting material by chemical or enzymatic
deacylation o f penicillin G. Some cephalosporins can be produced
using penicillin G as the starting material. The chemical synthesis
o f 6-APA is shown in the Fig. A - 7 . The enzymatic route is usually
accomplished using acylase enzymes bound to supports and operating
continuously in columns to convert the penicillin G to 6-APA. The
6-APA is converted by chemical acylation with appropriate acid chlo-
rides and results in some of the structures shown in Fig. A - 8 . Enzy-
matic methods are also available to convert the 6-APA to the semi-
synthetic penicillins, but such methods cannot compete economically
with the chemical conversion. Other semisynthetic penicillins such
as oxacillin, cloxacillin, dicloxacillin, and flucloxacillin also
exhibit lactamase inhibitory properties. Clavulanic acid (produced
along with cephalosporins by Streptomyces clavuligerus) and olivanic
acid (produced by Streptomyces olivaceus) are produced by fermentation
and are potent irreversible (suicide) inhibitors of B-lactamases but
have no or very weak antibiotic function on their own. They are used
in conjunction with one of the other semisynthetics such as amoxycil-
lin to give a much broader spectrum product so that the penicillin
81
ORNL DWC 88-238
PCI.
6-AMINOPENICILIANIC ACID
Fig. A-7. Chemical synthesis of 6-aminopenicilanic acid (6-APA). Source: G. J. M. Hersbach, C. P. van der Beek, and P. W. M. van Dijck, "The Penicillins: Properties, Biosynthesis, and Fermentation," pp 45-140 in Biotechnologv of Industrial Antibiotics ed E. J. Vandamme, Marcel Dekker, Inc., New York, 1986.
a2
NAME R -
CI
CI
CI
mCH3
6 OCH,
PROPICILLIN
OXACILLIN
CLOXACILLIN
DlCLOXAClLLlN
FLUCLOXACILLIN
ORNL OWG 00-240
M ETlCl LLI N
AMPICILLIN
AMOXYCILLIN
NH2
!/- CH - CAR BEN IC I LLI N
\/ COOH
Fig. A-8. Chemical structures of some semisynthetic penicillins. Source: G. 3. ?1. Hershmh, C. P. ~lan der Beek, and P. W. M. van Dijck, "The Penicillins : Properties, Biosynthesis, and Fermentation, " pp 45-140 in Biotechnology of Industrial Antibiotics ed E. J . Vandamme, Marcel Dekker, Inc., New York, 1986.
-
8 3
is effective even against bacteria which are normally resistant to
the j3-lactarn antibiotics such as certain resistant Gram-positive
bacteria and Gram-negative bacteria including Pseudomonas s p .
Penicillin G can also be converted to 7-aminodeacetoxycepha-
losporanic acid (7 -ADCA) in about a 70% yield by oxidation to its
sulfoxide, conversion of the sulfoxide to the corresponding deacetoxy-
cephalosporanic acid with transient protection of the carboxyl
group, and subsequent deacylation as shown in Fig. A - 9 . The semisyn-
thetic cephalosporins which can be formed by this route are shown in
Fig. A - 1 0 .
84
ORNL DWG 88-252
0 I I
CH2- C PENICILLIN G NHlZ2c;: 0 //c \
0 OH
I o
0 I
(CH,),SiN = C I - 0 - SI(CH3) I CH3
PYRIDINE HBr 80%
>90% PCI 5
I ROH
0 II
CH2- C, + 0 OH
7-AMINODEACETOXYCEPHALOSPORANIC ACID OeC 'OH
Fig. A - 9 . Conversion of penicillin G into 7-aminodeacetoxy- cephalosporanic acid (7-ADCA). Source: G . J. M. Hersbach, C . P. van der Beek, and P. W. M. van Dijck, "The Penicillins: Properties, Biosynthesis, and Fermentation," pp 45-140 in Biotechnology of Industrial Antibiotics ed E. J. Vandamme, Marcel Dekker, Inc., New
-
Y c r k , 1385.
85
ORNL DWG 88-244
0 /--/ 1 1
CH3 - CH2 -N, N-C -NH
I
k-- d 0 0
OH CH3
CEFOPERAZONE
OH 0
I CH3
CEFAM AN DOLE
CEFADROXIL
I CH,CH, -
CEFOTIAM
, CH3 N
CEPHRADINE
S - i,!N N
I
NH T-('
0 -'JCH2 -
C O" 'OH
H
CE FATRlZl NE
Fig. A-10. Chemical structures of some semisynthetic cephalo- sporins. Source: G. J. M. Hersbach, C . P. van der Beek, and P. W. M. van Dijck, "The Penicillins: Properties, Biosynthesis, and
ed E. J. Vandamme, Marcel Dekker, Inc., New York, 1986. Fsri i ientat ion," pp LtJ-ILtu I C 1 1 . n in S i o t e c h n o l o ~ v o f Indus t r ia l Antibiotics
APPENDIX B
Production of Cephalosporin Antibiotics
89
Appendix B. PRODUCTION OF CEPHALOSPORIN ANTIBIOTICS
Cephalosporins are produced by a fermentation process which is
very similar to that used in the production of penicillin. The dif-
ferences are in the growth media, microorganism used and the final
fermentation broth concentration of the antibiotic. Table B-1 lists
four typical growth media for cephalosporin fermentations taken from
the patent 1iterature.l' The production .media is a complex media
with corn steep liquor, meat meal, sucrose, glucose, and ammonium
acetate used. Several cephalosporin producing organisms (C-2237 and
C-3009) can produce the antibiotic while growing on a paraffin-based
growth media as shown in Table B - 2 . The fermenter is normally
operated at 22°C and a pH of 6.9 (5 to 7.3) with an air rate of 0.8
to 1.2 volumes per volume of medium per minute. The overall fermen-
tation takes about 114 hours. Figures B-1 and B-2 give the chemical
structure of some clinically important cephalosporins while Tables B-
3 and B-4 provide information on the clinical activity of the
cephalosporins against a wide variety of organisms.
Cephalexin was one of the first cephalosporins produced and i s
one of the most often used. It has now come off of patent restric-
tions and is available in generic form. Cephalexin is produced by
chemically splitting of the cephalosporin C to make 7-minocephalo-
sporanic acid (7-ACA) and then a chemical reacylation (see Fig. B-3).
Cephalosporins are produced by classes of microorganisms known
as fungi and actinomycetes. Within these classifications, a large
number of strains have been found which produce the antibiotic, and
Table B - 5 provides a listing of some of these organisms and the par-
ticular cephalosporins which they produce. Unlike the penicillin
fermentation, the final fermentation broth concentration is quite
low, 0.8 to 4 .0 g/L depending upon the particular cephalosporin pro-
duced and the organism employed."
Structurally, the cephalosporins are very similar to the peni-
cillins and several are being produced commercially from penicillin
G by biosynthetic means. Figures B-2 and B-3 illustrate the general
pathway. One advantage of this process is that the penicillins can
90
Table B-1. Cephalosporin production mediaa
Percent Component Medium A Medium B Medium C Medium D
Peanut meal 4 . 0 4 . 0 4 . 0 4 . 0
Soybean meal 2 .0 2.0 2 . 0 2 . 0
Beet molasses 3.45 3 .45 3 .45 3 .45
Methyl oleate 0 . 5 7 5 0 .575 0 . 5 7 5 0 .575
Lard oilb 6 . 0 6.0 6 . 0 6 . 0
_ _ - - - - Sodium sulfate 0 . 4 0 . 3
Ammonium sulfate - - - _ - - - - - 0 . 1
_ _ _ 0 . 4 Methionine - - - - - -
_ _ _ Calcium carbonate 0 . 2 0.2 0.2
0 . 4 Calcium sulfate - - - _ _ _ - - -
Yield: pg Cephalosporin/ml 3240 3560 4190 3920
aD. A . Preston and W. E. Wick, "Laboratory Evaluation of Cephalo- sporin Antibiotics," DeveloDments in Industrial Microbiologv Volume 16, American Institute of Biological Sciences, Washington, D.C. ( 1 9 7 5 ) .
bLard oil is rarely used now as it causes problems in the separation and purification steps; continuous carbohydrate feed is now commonly used.
91
Table B-2. Carbon utilization for cephalosporin productiona
Strainb Carbon Source Conc. % Growth' Cephalosporins produced d
(Pg/ml)
C-2237 n-Paraffine 8 +++ 120 25 110 5 Sodium acetatef 5 U 25 10 Soy bean oil 5 +++ 15
G 1 yce ro 1 5 +++ 85 5 60 Glucose 8 U+ 160 35 120
- - _ _
C-3009 n-Paraffind 8 +++ 18 3 15 5 Ethanol 5 +++ 15 - -
15 Sodium acetatee 5 U lo 5 Soy bean oil 5 +++ 6 5 Glycerol 5 +U 8
Glucose 8 +++ 50 1 5 30
aD. A. Preston and W. E. Wick, "Laboratory Evaluation of Cephalo- sporin Antibiotics," DeveloDrnents in Industrial Microbiologv Volume l6, American Institute of Biological Sciences, Washington, D.C. (1975).
- - - - - -
bP. persicinus C-3009 and P. carneus C-2237
cSymbols; +++, very good; u, good; +, poor growth.
dDACPC-Deacetoxycephalosporin C ; DCPC-Deacetylcephalosporin C ; CPC-Cephalosporin C.
eComponent; C12-22.3%, C13=49.9%, C14=27.5%, C15=0.4%
fA mixture of one part acetic acid and two parts sodium acetate. A 2% mixture in the medium and 1% fed at 72, 96, and 120 hours.
9 2
ORNL DWC 00-270
ONa
CEPHALOTHIN SODIUM
O H H H H I, I I I , s /
N=N
I / 1 , N=C ’ ‘ N - c H Z - c - N - c c c - H
,C-N\ C 0 C c \CH, - S -6
I ’s ’ ’CHS
ONo
CEFAZOLIN SODIUM
CEPHALORIDINE
CEPHRADINE
o”c‘oo CEPHALEXIN
0 H CHZO H 7 /I I I ,S\ 1
-- - ’ L C H - C - N - C - C C - H ’ S I 1 I 0
,C-N, c II 0 C ” ‘CH2- 0 - C - NH2
I
OH
CEFOXlTlN
H O H H H I 1 I S
,-
’ , i C - C N C C C H 0
C N C, I
ONHS 0’ C CH, 0 C CHs
CEPHALOGLYCIN
Fig. B-1. Structures of many of the cephalosporin antibiotics. Source: D. A. Preston and W. E. Wick, “Laboratory Evaluation of Cephalosporin Antibiotics,“ Developments in Industrial Microbiolom, 16. American Institute of Biological Sciences, Washington, D.C. (1975).
93
ORNL DWG 88-24!
L-a -AMINOADIPIC ACID ACTIVATED L- a -AYINOADIPIC ACID HO "2 /CHJ
\ C - C - C H
\ 0 d II 4 0
D
/ I HO NH2
- C - CH- (CH2)s - C - N - CH- CHI - SH
02\ H20 OH
d -(L- a -AMINOADIPYL)-L-CYSTEINE
0 4H
II 0 -- t SH
\\ ___c c - CH- ( c H ~ ) ~ - c - NH- cH- cL, C?S / I
no NH,
d -(L- a -AMINOAOIPYL)-L-CYSTEINYL-D-VALINE
\ OH
ISOPENICILLIN N 0
OH
PENlCLLlN N
0 0 0 2 HZN\ II HZN\ - C - (CH2)s - 21 - NH-C-
7 ,', -L- - (CH2)3 - C - N H - - - ~ " , , -
O* OH 0' ' h A C H , OH 0 'r CH20H
_ _ .- C
C O g OH o//
\
OH
DEACETYLCEPHALOSPORIN C OEACETOXCEPHALOSPORIN C
ACETYL CoA 0 ,s. c - (CH2)s c NH f 1 1 - -L - 0
I 0 N\, I-CH, 0 - CHs
C 0 OH
CEPHALOSPORIN C
Fig. B-2. Biosynthetic pathway for the formation of cephalosporin.
9 4
Table B-3. Clinical characteristics of common cephalosporin antibioticsa
Cephalothin Cephaloridine Cephaloglycin Cephalexin Cefazol in b Charac te r i s t i c
In v i t r o a c t i v i t y
aga ins t gram+ cocc i (except group D s t r e p ) +++
In v i t r o a c t i v i t y
aga ins t cephalospor inase- nega t ive gram negC tt
I n v i t r o a c t i v i t y
aga ins t cephalospor inase-
producing gram neg d
I n v i t r o a c t i v i t y
aga ins t Pseudomonas
fttt +++ tt
+++ ++++
Route of admin i s t r a t ion Pa ren te ra l Pa ren te ra l
R e l a t i v e peak concen-
t r a t i o n i n mouse blood a f t e r 20 mgJkg ++ +tt
R e l a t i v e d u r a t i o n + +tt
+
_ -
Oral
++
ft
++
Ora l P a r e n t e r a l
+ +*+ t+++
++ ++ ++*
aD. A. Pres ton and W. E . Wick, "Laboratory Evaluation o f Cephalosporin A n t i b i o t i c s , " Developnents i n I n d u s t r i a l Microbiology Volume 16, American I n s t i t u t e of B io log ica l Sc iences ,
Washington, D.C . (1975).
bSymbols: ++++ = very e f f e c t i v e ; tt+ = e f f e c t i v e ; ++ = moderately e f f e c t i v e : + = l i t t l e e f f e c t ; f = marginal: -- = no e f f e c t .
' e . g . , E . c o l i , Pro teus m i r a b i l i s , K l e b s i e l l a rmeumoniae. Salmonella s p . . S h i g e l l a s p .
d e . g . , Indole-producing Proteus s p . , Enterobac ter sp., S e r r a t i a s p .
95
Table B-4. Characteristics of cephalosporins of clinical interest
~
Specific activity (minimum inhibitory concentration, pg/ml)
Method of administration against the following: Name Injection Oral S. aureus Pseudomonas
Cefamandole + 0.9 0.8
Cefazol in + 0.8 2
Ce f oxi t in + Cephalexin
Cephaloglycin
+ +
2 6
5 9
2 2
Cephaloridine + 4 9
Cephalothin + 0 . 4 17
Cephap irin + 0.6 13
Cephace trile + 2 a
Cephradine + 5 20
96
ORNL DWC 88-243
(CH3)3SICI R1- C - NH x(J - R1-c-NHnJ O O / CH2-R2 BASE 0 / CH2-R, I
COOH COOSl( CH3)
R OH 2 PCI 1 1 L?JcH2-R2 COOSI(CH3)3
R , = 0 R,= - H
OR
H 0 I I1
R, = HOOC - C - (CH2)3 - R,= - 0 - C- CH3 I
CHZ
Fig. B-3. Chemical deacylation of cephalosporin C.
97
1 T a b l e B-5. Microorganisms which produce various cephalosporins
Compound Microorganism
Cephalosporin C
Deacetylcephalosporin C
Deacetoxycephalosporin C
A 16886 A
A 16884 A
Cepharrycin C or A16886 B
Cephamycin B
Cephamycin A
F1
c2
Penicillin N
Cephalosporium acremonium, C. polyaleunn, Arachnomyces minims, Anixiopsis peruviana, Spiroidium fuscum, Paeciiomyces persicinus, Paec. carneus.
Mutants of C. acrenwnium, C. polyaleurum, Ar. minims, An. peruviana, Sp. fuscum, Paec. persicinus, Paec . carneus
Mutants of C. acrezzwnium, C. polyaleurum, At-. minims, An. peruviana, Sp. fuscum,
Emericellopsis sp., Paec. persicinus, Paec . carneus, Diheterospora chlanydosporia, Scopulariopsis sp., Streptoqces lipmii, Str. clavuligerus
c. chrysogenum, e. sp.,
Str. Clavuligerus
Str. lipmanii
Str. clavuligerus, Str. lactamdurans
Str. griseus, Str. chartreusis, Str. cimamnensis, Str. fimbriatus, Str. halstedii, Str. rochei, Str. viridochromgenes
Same as Cephamycin B producers.
Mutants of C. acremnium
Mutnats of C. acrenwnium
A l l strains which produce cephalosporins
'Source: T. Kanzaki and Y. F'ujisawa, "Recent Progress in Cephalosporin Fermentations,"J. Takeda. Res. Lab. 2 (2) 324 (1975).
98
Table B-5. (Continued)
6-Aminopenicillanic acid C. acremonium
Tripeptide P 3 C . acremonium (6-(L-a-aminoadipyl)-L-cysteinyl-D-valine)
Tetrapeptide P 2 C. acremonium (a-aminoadipic acid, cystein, valine and glycine)
Tetrapeptide P 1 C. acremonium (a-aminoadipic acid, cysteine, /I-hydroxyvaline, and glycine)
Peptide S 1 Mutants of C. acremonium (Dimer of 6-(L-a-aminoadipyl)-L-cysteinyl-D-valine)
Peptide S 2 Mutants of C. acremonium (Disulfide of 6-(L-a-aminoadipyl)-L-cysteinyl-D-valine and methanthiol)
99
be produced at a lower cost than the cephalosporins. Cephalosporins
can also be produced chemically; however, the large-scale commerciali-
zation of the process is not feasible because of the operating con-
ditions. Some steps require temperatures of - 7 4 " C , while other steps
occur at 120°C, and expensive and toxic chemicals are required (such
as benzene, toluene, lead tetraacetate, phosgene, pyridine, methylene
chloride, L-(+)cysteine (precursor), t-butanol, and sodium acetate).
However, all of these compounds can be found in quantity at many
university chemistry departments and could be used to serve the
needs of a small area. The step-by-step syntheses is given by
Woodward et ~ t 1 . 4 ~ and is available in any chemistry department
library. A mathematical model for the fermentation of cephalosporin
C is given by Matsumura et a1.46
Product recovery
Cephalosporins are extracellular fermentation products, allowing
a bulk filtration to be used to separate the solid matter from the
antibiotic containing fermentation broth. Recovery from the filtrate
is difficult due to the low concentration of product and the need to
remove high molecular weight (1000 MW) biological compounds which
can lead to allergic and toxic manifestations when the drug is
administered.40 This is not as important for the oral product as
for the products for injection. Many different separation and puri-
fication schemes are employed, including conventional solvent extrac-
tion, ion exchange resins, liquid chromatography with conventional
resins and salting out procedures. Fig. B - 4 gives a general process
flow sheet for the recovery and purification of cephalosporins. The
general procedure is to filter the fermentation broth at acidic pH
(5 .O) , followed by adsorption of the filtrate on activated carbon,
removal of the adsorbed antibiotic by contacting the carbon with a
mixture of water and a polar organic solvent, contacting the eluate
with an anion exchange resin, and eluting the resin with a salt
solution at a pH of from 5.5-10.0. Frequently, the carbon adsorp-
tion steps are replaced with a precipitation step. Many different
precipitations are possible. Among the more common are4'
100
CONCENTRATE
ORNL D W G 88-233
CONCENTRATE CON
MEMBRANE -h FILTER ULTRA FILTER REVERSE OSMOSIS
CONCENTRATE FERMENTER CONCENTRATE CONCENTRATE
I
CHILLED WATER
PERM E AT E _____)
BYPASS REVERSE OSMOSIS MODULE
D PERMEATE
:ENTRATE
CELL HARVEST PROTEIN, CELL WASHING PIGMENT REMOVAL n
U HPLC PURIFIED I
ANTIBIOTIC CEPHALOSPORIN C. RECOVERY PROCESS FROM FERMENTATION BROTH
CONCENTRATE RETURN
REVERSE OSMOSIS CONCENTRATION OF CEPHALOSPORIN C
Fig. B-4. Schematics for recovery and concentration of cephalo- sporin C. Source: M. Kalyanpur, W. Skea and M. Siwak, "Isolation of Cephalosporin C from Fermentations Broths Using Membrane Systems and High-Performance Liquid Chromatography," DeveloDments in Industrial Microbiology, 26 455 (1985).
101
Crystallization of the potassium or sodium salt from purified aqueous solution of the cephalosporin by concen- tration and/or addition of large volumes of a miscible solvent.
The zinc salt (also copper, nickel, lead, cadmium, cobalt, iron, and manganese) can be crystallized from purified aqueous solutions.
Insoluble derivatives such as the N-2,4 dichlorobenzoyl cephalosporin and tetrabromocarboxybenzoyl cephalosporin are crystallized as the acid from aqueous solutions.
4 . Sodium-2-ethyl hexanoate will precipitate the sodium salt of N-derivatized cephalosporins from solvents.
Figure B-5 gives a typical flow diagram for the purification of cephalosporins using ion exchange resins.
102
CEPHALOSPORIN C BROTH
c ANION-
EXCHANGE ANION-
EXCHANGE RESIN RESIN
c IRA 94 - IRA 6 8
CATION- (EXCHANGE H'FOR EXHANGE INORGANIC CATIONS) RESIN
IRC a4
ORNL DWG 8 8 - 2 4 2
CEPHALOSPORIN C ADSORPTION
20% ACETONE
CEPHALOSPORIN C
NON-IONIC RESIN XAD 2
HP-20
ELUATE
ELUATE
PYRIOYLACETATE
ANION- EXCHANGE
RESIN IRA 6 8
I ELUATE TO ELUATE
I ELUATE *
NON-IONIC RESIN ADSORPTION
ANION-EXCHANGE ADSORPTION
Fig. B-5. Cephalosporin C purification f r m bro'h by adsorption. Source: M. E. Wildfeuer, "Approaches to Cephalosporin C Purification from Fermentation Broth," Purification of Fermentation Products, 156 (1985).
APPENDLX c
Production of Tetracycline Antibiotics
105
Appendix C. PRODUCTION OF TETRACYCLINE ANTIBIOTICS
Tetracycline antibiotics production utilizes preinoculation,
inoculation, and fermentation tanks similar to those of penicillin.
The processing scheme is shown in Fig. C-1, and the procedure is
shown in Fig. C-2. Oxygen must be provided for oxygen transfer and
aeration rates of 1 volume of air per volume of fermenter per minute
(WM) are customary. Oxygen enriched air provides faster growth and
higher yields. The fermenter can have a 100- to 150-d working
volume with three open turbines that are 1450 to 2100 mm in diameter
with maximum speeds of 80 rpm and a power input of 300 kW (400 Hp)
at the shaft (up to 3 kW/m3). Oxygen supply continuity is very
important because interruption of the oxygen supply for more than 10
min results in the stopping of tetracycline production.
Strains are kept either in a freeze-dried or liquid-nitrogen
state as spore stock. A culture is started by sporulation on an
agar slant, followed by the second sporulation in a stationary cul-
ture on millet in flasks. The first step requires 7 to 10 days, and
the second 14 days. About l o 5 t o l o6 spores/cm3 are inoculated into the first submerged culture fermentation in the preinoculation tank
and allowed to grow for 24 to 26 hours. The contents of the first
fermentation are transferred to the inoculation tank, allowed to
grow for 18 to 20 hr, and then this material (about 5 % of the fer-
mentation vessel volume) is added to the fermentation vessel with
the nutrients and allowed to grow for 100 to 200 hr with tetracycline
production. All growth takes place at 2 9 ° C .
The medium for tetracycline production includes sucrose, glucose
or molasses, 2.5% wt/vol, organic nitrogen sources (soybean meal or
corn-steep liquor, 1.7%), a buffer (calcium carbonate, 0.2 to 0 . 3 % ) ,
inorganic salts (NaC1 and KH2P0 , ) , and vegetable oil (0.2%). Chloride
ions serve as precursors of chlor-tetracycline, and benzylthiocyanate
is added as an inhibitor of undesirable metabolic pathways during
lack of oxygen. The culture is monitored for pH, residual sugar,
106
ORNL D W G 0 7 - 4 7 3
AMMONIUM SALT f-
1
Fig. C-1. Production scheme for oxytetracycline. D. J. D. Hockenhull, The Fermentation of the Tetracyclines, Source:
Interscience Publishers Inc., New York (1959).
107
Inoculum Preservation (spores on agar slant or in soil)
Agar Plates
Spores as Inoculum
Shake Flask
I 24 hr
2% Meat extract; 0.05% Asparagine; 1% Gulcose; 0.5% K2HP04; 1.3% Agar
2% Corn steep liquor (50% solids); 3% Sucrose; 0.5% CaC03
Prefermenter
5% Inoculum 19-24 hr pH 5.2-6.2
Same as for shake culture
2-10% Inoculum 60-65 hr 28 "C pH 5.8-6.0
Purification from the clear broth after removal of the mycelium
1% sucrose; 1% corn steep liquor; 0.2% (NH4)2P04; 0.2%KH2PO4; 0.1% CaC03;
0.005% ZnS04-7H20;
0.00033% MnC12-4H20
0.025% MgS04'7H20;
0.00033% CuS04*5H20
Fig. C-2. Production chart for chlortetracycline.
Source: D. J. D. Hockenhull, The Fermentation of the Tetracyclines, Interscience Publishers Inc., New York (1959).
108
respiration (CO, concentration), increase of biomass in concentration
and in volume, and morphology.
Nutrients are sterilized at 120°C for 40 min or at 140°C for 1
min with C and N sources sterilized separately. Aeration ranges up
to 0.8 medium vol/min and oxygen overpressure of up to 0.1 MPa are
used. The pH, oxygen content of the medium (at least 20% satu-
ration) and the CO, in the exhaust air are monitored continuously.
Product Recoverv and Purification
Isolation methods for tetracyclines must take into account
their amphoteric nature and the possibilities of their polymerization
or rearrangement. Several methods have been used to recover and
purify tetracyclines and some of these are listed below.”
1. Adsorption on diatomaceous earth or active charcoal with subse-
quent chromatography or selective extraction.
2. Extraction from acid or alkaline medium. The most frequently
used extraction agent is 1-butanol, owing to its suitable par-
tition coefficient and economic availability.
3 . Direct mash extraction based on solubilizing the antibiotic by
acidification, precipitation of Ca2+ with ammonium oxalate,
addition of quaternary ammonium compounds as carriers, and
extraction of the metabolite with an organic solvent, usually
one of the methylalkyl ketone type.
4 . Precipitation (dry salt) process based on precipitation the
antibiotic from dilute aqueous solution of aryl azosulfonic acid
dyes. Tetracyclines are precipitated as complexes with alkaline
earth metal compounds or with primary and secondary alkyl amines.
5. Solvent extraction of the antibiotic with salt, based on salting
out (NaC1) the antibiotic from the aqueous to the organic phase
(1-butanol). This method is also suited for refining a crude
product.
Oxytetracycline is purified by combining the release of oxytetra-
cycline into the medium by acidification, precipitation of ballast
compounds with K,Fe(CN), and ZnSO,, extraction of liquid fractions
109
with butyl acetate, and precipitation of Ca2+ with oxalic acid.
After adjusting the filtrate with EDTA, Na,SO,, and citric acid, the
crystalline Sase of oxytetracycline is obtained.
Further purification is carried out by crystallization as salts
(e.g., hydrochlorides) or bases. Particularly efficient is crystal-
lization from boiling solvents, such as lower alcohols, ketones, or
aliphatic ethers of ethylene glycol, which yield nonhygroscopic
preparations, Residual amounts of antibiotic in the mother liquor
are increased by oxalate and chloride anions, while sulfate anions
have the opposite effect. Crystallization is most efficiently per-
formed at 2 ° C for 3 hours.48
Semisynthetic tetracyclines are prepared from oxytetracycline by
chemical synthesis. Methacycline, doxycycline, rninocycline and roli-
tetracycline can be prepared. Methacycline is made by a three-step
synthesis and is isolated as a sulfosalicylate which is converted to
a hydrochloride of methacycline by crystallization from an acid mix-
ture of acetone and methanol in a 62% yield.
Analytical Methods
Chemical assays of tetracyclines based on metal complexes utilize
the characteristic absorption of fluorescence of the complex formed.
Using this method, one can analyze the principal tetracyclines both
in biological material and in commercial preparations. Polarographic
assay of tetracyclines is based on their reduction in a boric acid-
sodium borate buffer. During the fermentation and isolation process,
paper chromatography, thin-layer chromatography, and gel chromatog-
raphy can be used for assay. Recently, HPLC, using both regular and
reverse phase packings and ion exchange packings, has been used for
analyses of the tetracyclines.27
APPENDIX D
SulfonaInides
113
Appendix D. SULFONAHIDES
The sulfonamides (sulfa drugs) represent a large class of anti-
microbial drugs which are produced by chemical reaction. The sulfon-
amides came into common usage in the mid 1930's and are still produced
in quantity today. From the base compound, many clinically active
derivatives have been produced, and there are over 5,000 different
sulfonamide derivatives. Figure D-1 presents the chemical structure
of the most important sulfonamides. The processing steps necessary
to produce purified sulfonamides are common to the organic chemical
and dyestuffs industries and are described briefly below.16
The usual method of sulfonamide preparation is the reaction of
N-acetylsulfanilyl chloride (ASC) with the appropriate amine.
$ / c-c, B base S I c-C\ 0 CH, -C-NH-C, , C-;-Cl + RNH, - CH,-C-NH-C\ , C-S-NHR
c-c 0 c-c 0
Extra amine or base is used to neutralize the hydrochloric acid
freed in the reaction. The acetyl product is treated with aqueous
alkali to the free amino compound. ASC is obtained by chlorosulfo-
nation of acetanilide. The drugs are white powders melting above
15OoC, and azo and nitro derivatives may be yellow. The sulfas are
sparingly soluble is cold water but are somewhat soluble in alcohol
or acetone. Assays for the sulfa drugs are usually done by titration
with nitrous acid and paper or thin-layer chromatography.49
114
NAME
SULFANILAMIDE
SULFAPY RlDlN E
SULFATHIAZOLE
SULFADIAZINE
SULFACHLOROPYRADAZINE
SULFADIMETHOXINE
SULFAETHIDOLE
SULFAMETHAZINE
SULFAMETHAZOLE
ORNL DWG 88-275
STRUCTURE
H 2 N @ S O * - N H ~
H /
-- “0 /H -“I /H -“g
H / -“a CI
N-N
0 -CH3
H / - N
rsTC2H5 N-N
-N$3 CH3
H /
-NrsTcH3 N-N
-N?rH3 SULFAMETHOXAZOLE
N-0
Fig. D-1. Therapeutically important sulfa drugs and sulfones. Source: EncycloDedia of Chemical Technology, Third Edition, ed Kirk-Othmer, Interscience, New York (1978).
-.
11 5
NAME
SULFAMETHOXYPYRIDAZINE
SU LFAM ETER
SULFAMOXOLE
S U LFAPH EN AZOLE
SULFAPYRAZINE
SULFAQUINOXALINE
SU LFlSOM I DI N E
SULFISOXAZOLE
SU LFACYTIN E
ORNL DWG 88-276
STRUCTURE
H2N @ S 0 2 - N b OCH,
N-N'
- <@ OCH,
-Nb H N
/
0
116
NAME
SULFACETAMIDE
SULFAQUANIDINE
N’-ACETYLSULFISOXAZOLE
SUCCINYLSULFATHIAZOLE
PHTHALYLSULFATHIAZOLE
SALICYLAZOSULFAPYRIDINE
MAFENIDE
DAPSONE
ACEDAPSONE
PRONTlSlL
ORNL DWG 88-277
STRUCTURE
H3 Lyf H3
- N ‘C- CH3
0’’ .H
H --N@!502 - N /
0 v’p N
@’O: - H / N v’p N
k-J
/ CH2@ SO, - NH,
NH2
NH2
APPENDIX E
Bacitracin Production
1 1 9
Appendix E. BACITRACIN PRODUCTION
Bacitracin has been produced using several different types of
media. They include (1) synthetic media containing amino acids as
carbon and nitrogen sources, (2) rich media containing beef extracts
or tryptone broth, and ( 3 ) high-producing media with soybean meal and
starch or dextrines/sugars as nutrients. Bacitracin production
levels are on the order of 400 to 600 IU/ml where 74 IU (intemational
units) corresponds to 1 mg of the Second International Standard of
bacitracin or 5 to 9 g/liter.
The bacitracin-producing strains of Bacillus licheniformis are
kept on agar slants or in the spore form in dry sterile sand. The
bacitracin production process is shown in Fig. E - 1 and E - 2 . Fig. E -
3 shows the flow scheme and gives the media compositions used in the
various steps of the process. A suitable amount of the culture is
used to inoculate shake flasks of tryptone or peptone broth. An
intermediate fermenter is usually used as a seed tank for the main
fermenter. The fermenters are equipped with an agitator and a
jacket for steam or water. The medium is sterilized either by batch
or continuous sterilization. The fermentation process is usually
monitored by continuous pH and 0, measurements. The fermentation is
carried out at 30 to 3 7 " C , for 24 to 30 hr. Recently, bacitracin has
been produced by cells of B. licheniformis immobilized in polyacryl-
amide gel, but this is not commercial.14
Recovery and Purification of Bacitracin from Fermentation Broth
Bacitracin is prepared by direct precipitation form the clarified
fermentation broth by salts of divalent cations, primarily zinc, but
nickel, manganese and cobalt have also been used. Table E - 1 gives the
solubility of bacitracin, zinc bacitracin, and bacitracin F in various
solvents, and Table E-2 lists some precipitation agents for bacitra-
cin. Zinc bacitracin is used primarily as an animal feed-grade
product and is probably the most simple separation. After fermen-
tation, salts such as methylene disalicylic acid, lignin sulfonic
acid, hydroxymethane sulfonic acid, and alkylbenzene sulfonic acid
120
A - c STERILE AIR
SOY BEAN MEAL -
STARCH - -
I I SEED FERMENTER STEAM
00 MASH COOKER
- ___)
FERMENT E R c.$FzR MEDIA
ORNL DWG 88-283
00
SEED TANK
STERILE AIR
STERILE AIR
FERMENTER
BUTANOL
CAKE T O TREATMENT
SPENT BEER TO STRIPPER
CENTRIFUGAL INTERMEDIATE
BUTANOL VAPOR RECOVERY CONCENTRATOR
t BACITRACIN PRODUCT
Fig. E - 1 . Bacitracin production flow and equipment scheme. Source: 1, Second Edition, Academic Press, New York (1979).
H. J. Peppler and D. Perlman, Microbial TechnoloEy Volume
Y - h
121
I Inoculum Preservation 1 I (spores on agar slant I
Shake culture 4 X 1 L Erlenmeyer flask with 200 m l tryptone or
or peptone culture medium 18-24 hr I at 37°C
Same as for shake culture
6 hr at 37°C 1 with intensive
aeration
Prefermenter (3000 L) 5% Soy meal; 1.2% Sucrose; 0.2% (NH4),S04 ; 0.2% cam3
Growth to I log phase
5% Soy meal; 2.4% Sucrose;
J Production Fermenter 0.2% (NH4),SO4; 0.2% cam3
30 hr at 7, Furif ication a. For pharmaceutical uses: Extraction with n-wztanol, extraction of the organic phase with buffer, concentration of the aqueous phase, ion- exchange c-hromatography
b. For use in animal feeds: Spray drying of the whole femtation broth
Fig. E-2. Block diagram for the production of bacitracin.
122
Table E-1. Solubility of bacitracins in various solventsa
Solvent
Solubil i tyb (mg/ml )
Bacitracin Zinc Bacitracin Bacitracin F
Petrolether (bp 100-120°C) Petrolether (bp 40-60°C) Carbontetrachloride Toluene Diethylether Chloroform Pyridine 1 - Butanol Acetone Methanol Acetonitrile Water
- - 0.006 17.0 4.2 0.08
0.005 >40.
>40.
- - - -
0.009 _ -
0 . 0 0 2 0 . 0 7
0.68 0.06
0 . 0 0 5
16.1
1 7 . 3
10.1
0.002 0.007 0 . 3 7
_ - 0 . 2 4 11.4
>15.
>15. 0.09
0.07 11.2
a $3. Frayshov, "The Bacitracins: Properties, Biosynthesis, and Fermentation," pp 665-694 in Biotechnology of Industrial Antibiotics ed E. J. Vandamme, Marcel Dekker, Inc., New York, 1986.
bThe solubility of bacitracin (pharmaceutical grade) , zinc bacitracin (pharmaceutical grade), and bacitracin F (oxidized bacitracin) were tested at 25°C.
Table E-2. Precipitating agents for bacitracin.a
Ammonium molybdate Molybdic acid Ammonium rhodanilate Phosphomolybdic Azobenzene p-sulfonic acid Phosphotungstic Benzoic acid Picric acid Divalent metal ions (Zn, etc.) Picrolonic acid Furoic acid Salicylic acid Methylene disalicylic acid
acid acid
a $3. Fr$yshov, "The Bacitracins: Properties, Biosynthesis, and Fermentation," pp 665-694 in Biotechnology of Industrial Antibiotics ed E. J. Vandamme, Marcel Dekker, Inc., New York, 1986.
1 2 3
have been used for precipitation of the bacitracin salts. Bacitracin
can be adsorbed/precipitated on solid carriers like bentonite and
celite. After precipitation, bacitracin is concentrated by filtra-
tion, centrifugation, or evaporation, and then dried by drum or
spray drying.
For pharmaceutical-grade bacitracin, solvent extraction from the
clarified fermentation broth with butanol, or ion exchange followed
by solvent extraction is practiced. The precipitation of bacitracin
is accomplished with divalent metal ions, primarily zinc.
Assay methods for bacitracin include microbiological (in which
inhibition of a test organism is compared to a standard bacitracin),
paper and thin-layer chromatography, electrophoresis, isoelectric
focusing, and pulse polarographic techniques. The assay method of
choice is HPLC using reversed-phase packing and programmed gradient
elution or an isocratic system. A chromatogram is shown in Fig. E -
4 . HPLC has been used for monitoring the fermentation broth during
fermentation and during the purification process.50
m
r
C
-I 0
z
C 0
r
C 3
m
I
UV
AB
SOR
BA
NC
E (2
54 n
m)
SU
BS
TITU
TED
>
82 -B
'1 B
AC
ITR
AC
INS
BA
CIT
RA
CIN
A
OX
IDIZ
ED
BA
CIT
RA
CIN
A
APPENDIX F
Organic Product Fermentations
127
Appendix F. ORGANIC PRODUCT FERMENTATIONS
Ethanol process
The process for production of fermentation ethanol is similar
in its initial steps to antibiotic production in that a stirred-tank
batch fermenter (similar to Fig. F-1) is used to produce a fermenta-
tion product which must be separated and purified before use (Figs.
F-2, 3 , and 4 ) . The ethanol fermentation is fundamentally differ-
ent, though, because it is essentially an anaerobic process. Some
oxygen is required for proper growth, and the aeration equipment
may be in place in the fermenters to allow for the sparging that is
required for an antibiotic fermentation. The heat exchange and agi-
tation equipment may not be adequate for full- scale antibiotic fer-
mentation, because during an ethanol fermentation, broth viscosity
changes very little, and fermentations occur at a higher temperature
(35°C vs 23 to 25°C). Separation/purification equipment will gener-
ally not be adequate because the ethanol process only includes fil-
tration and distillation and azeotropic distillation (benzene)
equipment. Some distillation towers could possibly be used as
extraction columns to obtain some separation.
Organic acids
Acetic acid is now often produced in a submerged-culture batch
fermentation using a stainless steel fermenter with a high-speed
agitator on the bottom of the fermenter for high efficiency oxygen
transfer. The vinegar product is filtered using filter aids and
fining agents for purification. I n the submerged-culture citric
acid process, aeration and agitation requirements are similar to antibiotic production needs, and glass- or plastic-coated or 316
stainless steel fermenters are used (adaptable to sterilization and
antibiotics fermentations). The purification steps include filtra-
tion, precipitation, activated carbon and ion exchange columns,
crystallization, and centrifugation (which readily adapt to antibi-
otic purification needs). Gluconic acid is also produced in a sub-
merged fermentation at 30°C using air sparging in batch mode.
128
ORNL D W G 88-296
co
FOAM
lOLlNG C
MOTOR DRIVE
////// l///~/,//,///////,/!///\///////,/,////,
I
BAFFLE PLATES
SPARGER STERILE AIR INLET
FLAT-BLADE TURBINES
Fig. F - 1 . production. Source: Biochemical Enpineering, Academic Press, Inc., New York (1973).
Common stirred-tank batch fermenter for ethanol S. Aiba, A. E. Humphrey, and N. F. Millis,
129
ORNL D W G 88-293
ENZYME REACTOR
H 2 S 0 4 . W A T E R
C O R N S T O V E R 1 b L i , A C I D P R E T R E A T M E N T
I
S U Q A R S
I
t EXCHANGER
< a 0 <
- STERILIZER
MINERALS, V ITAMINS
YEAST
l i
R E S D U A I SOLIDS
CENT R IF U Q E
FERMENTER E N Z Y M E PRODUCTION
41
FILTER
S O L W S n ETHANOL
PROD UCTlON ADSORPTION OR ION E X C H A N Q E
AN A E R 0 B IC WASTE D lQ E ST 10 N
Fig. F-2. Equipment used in the production of ethanol from corn stover. Source: B. Atkinson and F. Mavituna, Biochemical Engineering and Biotechnology Handbook, Nature Press, London (1983).
130
FUSEL OILS
t . 25% ETHANOL ~
ORNL D W G 88-301
FUSEL OILS -
96%
12.5% ETHANOL 75.1% BENZENE a
U * 96% ETHANOL DECANTER
131
ORNL D W G 88-300
* -
RECYCLE I
Fig. F-4. Purification equipment for anhydrous ethanol produc- tion. Source: B. Atkinson and F. Mavituna, Biochemical Enpineering and Biotechnologv Handbook, Nature Press, London (1983).
132
Purification equipment includes filtration, evaporation, crystalli-
zation, centrifugation, and drying. Extracellular polvsaccharides
such as xanthan gum, dextran, and Zanflo, are produced in batch fer-
mentations followed by precipitation, solvent extraction, and combi-
nation of these. The fermentations require oxygen, heavy duty agi-
tation due to the high viscosity of the broth as the fermentation
proceeds, but very little heat exchange. The fermentation of micro-
organisms utilizes stirred, jacketed, sparged fermenters, followed
by centrifugation, extrusion, drying, and leaching. Cyanocobalamin
(vitamin B - 1 2 ) is produced in a submerged, aerobic, stirred fermen-
tation followed by high-speed centrifugation, heat treatment to
release the product, ion exchange, and activated carbon adsorption.
Another separation method uses an extraction with phenol or other
solvent and precipitation or crystallization. Riboflavin (vitamin
B-2) is produced by fermentation performed at about 29°C with agita-
tion and aeration in a jacketed fermenter. The product is recovered
by heating the broth followed by centrifugation, chemical reaction,
and filtration or adsorption and elution (all very adaptable and
necessary for most antibiotic production procedures). Glutamic acid
is another organic acid produced by fermentation, which is very
oxygen-transfer-dependent. The fermentation of glutamic acid and
penicillin are very similar, and both fermentations can and do use
the same fermentation and product-recovery equipment.
APPENDIX G
Enzyme Production Technology
135
Appendix G. ENZYME PRODUCTION TECHPiOLOGY
Enzyme production is based on the fermentation of a micro-
organism to produce the complex enzyme which is then purified by
various processing steps to yield a stable preparation. Semisolid
or liquid media are used, but only the liquid submerged process is
applicable for the discussion of antibiotic production in enzyme
equipment. The process flowsheets shown in Figs. G-1, G-2, and G - 3
detail the various equipment including the fermenter and associated
culture starting tanks, sterilization equipment, feed tanks, sterile
air supply, and the filtration and purification equipment for differ-
ent enzymes. The separation and purification equipment is very much
the same as that found in an antibiotic production facility, and
very little change would be required for the plant to produce certain
of the antibiotics on short notice. The basic differences are in the
various solvent extraction requirements and equipment, which in an
enzyme process involve only a single- or two-stage extraction, com-
pared to the multiple-stage, multiple-solvent processing required
for most antibiotic production schemes. EnzyKes are usually 1 to 2
orders of magnitude larger than antibiotics, giving rise to the dif-
ferent separation schemes used for enzymes such as ultrafiltration,
chromatography, and dialysis.
I36
ORNL DWQ 88-291
FERMENTER
C EN TR IF UQE
FILTER ENZYME SOLUTION
C IP IT AT IO N
SOLVENT
SOLVE NT PREC IPlTAT IO N
CHROMATOGRAPHY
CENTRIFUGE
FINAL PRECIPITATION VACUUM
ENDOCELLULAR ENZYME PRODUCT
Fig. G-1, Intracellular er?zFe purificatio.. flow sheet. Source: B. Atkinson and F. Mavituna, Biochemical Engineering and Biotechnology Handbook, Nature Press, London (1983).
1 3 7
ORNL DWG 88-290
FERMENTER
EXCHANQER HEAT h 1 c o o L ~ ~ ~ STORAGE PRrTREATMENT f
!\ -1 'Lp 1 DECANTER 1 60
___)
EVAPORATOR
HEAT EXCHANGER
E C IPlT AT10 N
FILTER PRESS
BACTERIAL FILTER
SPRAY DRIER1
'I PRODUCT
Fig. 6-2. Alternate enzyme recovery processes. Source: B. Atkinson and F. Mavituna, Biochemical Engineerinv and Biotechnology Handbook, Nature Press, London (1983).
138
- -
SEPARATION I P U R IF IC A T I 0 N
F I L T E R PRESS 11 PLANT
EXUDATE = ACETONE, E T H A N O L
ORNL D W Q 8 8 - 2 9 2
I t MICROBIAL ENZYMES
r--+
I I IFERMENTER, I ~ R T I-+- INGR E DIE N T S
r h VACUUM
FREEZE DRIER
DRY CRUDE P R O D U C T S
I T CENTRIFUGE 1 8
T R A Y DRIER wuu
E V A P O R A T O R - H E A T EXCHANGER
W
S A L T
FR A CT lON A P R O D U C T S
S T A N D ARDlZE D PRODUCTS LIQUID PRODUCTS DRY P R O D U C T S
DIL UT E Lla ID C 0 N C E N T R A T E D
TED
Fig. G-3. Comon commercial enzpe process. Source: B. Atkinson and F. Mavituna, Biochemical Engineering & Biotechnology Handbook, Nature Press, London (1983).
APPENDIX H
Single C e l l Protein Production
141
Appendix H . SINGLE-CELL PROTEIN PRODUCTION
Single- cell proteins are various groups of microorganism that
have been considered for food or feed use, including algae, bacteria,
yeasts, molds, and higher fungi. The dried cells of these organisms
are collectively referred to as single-cell proteins (SCP). In the
early 1 9 6 0 s , many major oil companies began researching the possibil-
i t y of using microorganisms to upgrade refinery streams with the aim
of reducing the cost of refining. Certain groups of microorganisms
are capable of selectively consuming specific components from these
streams which could then be returned to the refinery for further
processing. The resulting protein-rich biomass (yeast and bacterial
cells) might serve as a source of animal feed or even as a potential
feed source to supplement agriculturally produced human foods.
The Pure Culture Products SCP plant in Hutchinson, Minnesota,
produces 15.0 million lbs/yr of SCP for human consumption. The
plant has FDA approvals for the product (torula yeast, Candida
u t i l i s ) and utilizes a pure culture continuous fermentation under
sterile conditions. The entire plant operates under aseptic condi-
tions with facilities for steam sterilization and clean-in-place
(CIP). The main components of the plant consist of facilities for
raw materials storage and preparation, nutrient sterilization, fer-
mentation with two 20,000-gal continuous pressurized state-of-the-
art fermenters, facilities for air compression and sterilization, a
chilled water system for fermenter and product cooling, and a sepa-
ration and product recovery system. Figure H-l presents a block
diagram of this manufacturing facility.
The chilled water system utilizes 10°C-year-round well water
for a major portion of the cooling duty, thus saving a great deal of
energy. The heat transfer facilities consist of the fermenter cool-
ing baffles, plate and frame heat exchangers for the cooling system,
and tri-pipe exchangers for the pasteurization system. The separation
system uses centrifuges to separate the microorganisms from the fer-
mentation broth and a large spray dryer for drying the final pro-
duct. In addition a 540-unit Romicon ultrafiltration unit is in
142
STERILIZATION WASTE -
ORNL DWG 88-745
ULTRAFILTRATION UNIT
L
INOCULUM SYSTEM 1
10°C WELL CHILLED WATER WATER
COMPRESSED ,-+I - I- AGITATED PRODUCT SEPARATION U
2 e FERMENTERS STORAGE (CENTRIFUGES) cT w I - . (I)
-* N A ERA T E D - COOLING c SYSTEM
3O0C, 2 atm
4 RECYCLE
Fig. H-1. Block diagram of the Hutchinson. facility.
PASTEUR IZ A TI0 N
SPRAY DRYER
DRIED PRODUCT
MN, SCP production
143
place for the recovery of macromolecules (like antibiotics) from the
autolyzed fermentation broth.
The fermentation system operates under highly aerobic conditions
with hydraulic drives and turbine agitators to ensure high oxygen
transfer rates. The process is highly exothermic and operates at 30
" C with 1 0 ° C chilled water circulation for temperature control. The
fermenter is sterilized with 1 2 1 " C , 15-psig steam and has operated
for up to 75 days under aseptic conditions while maintaining a pure
culture. The facility can a l s o be operated in a batch mode as
required for various antibiotics production processes.
The Hutchinson plant was designed to also serve as a research
facility and thus has a complete fully equipped laboratory with
state-of-the-art analytical capabilities that would be required for
antibiotic production. The plant is adjacent to a small river
which would facilitate waste removal. The surrounding farming area
would be capable of providing nost of the raw materials required for
antibiotic production.
APPENDIX I
Inoculum Preservation
147
Appendix I. INOCULUM PRESERVATION
The preservation of production strains of antibiotic-producing
organisms over a long period is a basic requirement for a practical
fermentation. Not only their survival but also their capability for
product formation must by preserved. High-yielding strains have
often been damaged in primary metabolism during the strain selection
process, and they frequently degenerate during successive transfers.
The objective of preservation is to maintain strains as long as
possible without cell division. The three most common techniques
for preservation are low temperature (2 to 6 " C ) , frozen (-18°C or-
8 0 ° C in freezers o r - 1 9 6 ° C under liquid nitrogen), and lyophilization
(freeze-drying) with the addition of protective agents such as skim
milk or sucrose. Lyophilization is the preferred method for long-
term storage. 2 o
The preserved culture is revived by growth in a shake flask
liquid culture or on solid medium for spore formation. Standard
growth times for cultures are
Lyophilized cultures 4-10 days
Frozen cultures
Bacteria 4-48 hours
Actinomycetes 1-5 days
Fungi 1-7 days
Refrigerated cultures
Bacteria 4 - 2 4 hours
Actinomycetes 1 - 3 days
Fungi 1-5 days
Following the first flask culture, a second series of shake cultures
is made in more flasks from the first flask. For a spore crop, the
culture can be cultivated in specially devised liquid media, on
agar, or on a solid substrate in 2- to 10-liter glass vessels for 8
to 24 days on bran, peat, rice, or barley, with sterile aeration and
daily shaking. The total spore culture is suspended with a surface-
148
@ active agent like Tween 80 and transferred to the fermenter. Then,
depending on the volume of the production fermenter, fermenter pre-
cultures must be made. During all steps, the nutrient medium, tem-
perature of growth, inoculum age, and induction or repression
phenomena must be controlled for optimum production. Culture trans-
fers to the larger fermenters should be made while the culture is in
the log growth phase to minimize culture lag time at the next stage.
Simple apparatus are available for preservation of cultures by
freeze drying, from companies like Virtus , HETO, Labconco, and
Stokes Div. of Pennwalt. Figure 1-1 shows the survival of a culture
of of Mycobacterium tuberculosis var. BCG versus the temperature of
the freeze drying. The temperature vs time profile is important to
minimize damage to the cells in both the freeze and thaw processes.
The freezing rate should be about l"C/min down to -2O"C, after which
the rate should be as fast as possible down to the storage temperature
which should be below -55°C. The freeze drying should take place at
a constant temperature below - 3 0 ° C . Thawing should be as rapid as
possible. The cultures should be kept down to small volumes and in
containers which allow for rapid heat transfer, (2.0-ml thin glass
ampules are recommended). The electrolyte content of the suspending
medium should be kept low. The addition of glycerol (10 to 20%),
glucose (10%) or sucrose (10%) to the suspending medium will aid
cell survival. Temperature fluctuations should be avoided during
storage. If the storage temperature is permitted to rise and fall
above -2O"C, the cells will suffer extensive damage.
149
I I I
- - - /* -
-
-
70
60 - 50 de Y
2 40 2 > 30 a 3
20
10
0
TEMPERATURE (OC) Fig. 1-1. The effect of drying temperature on survival of freeze dried c e l l s . Source: P. W. Muggleton, "The Preservation of Cultures," Progress in Industrial Microbiologv, 4 191 (1962).
APPENDIX J
Ster i l i za t ion
153
Appendix J. STERILIZATION
This appendix will discuss three areas of sterilization which are
vital to the successful operation of an antibiotic plant-the steri-
lization of air, medium, and process equipment.
Sterilization of air implies the complete removal or destruction
of all viable particles ranging in size down to the smallest bacterial
cell, including bacteriophage and large viruses (0.5-micron). Only
three methods have been applied to the sterilization of air in com-
mercial fermentation processes. These are heat, filtration through
fibrous media, absolute filter media, and granular media. The most
commonly used method t o clean intake sir is filtration through beds
of fibrous material such as glass wool and slag wool.
Figure J - 1 shows the general process flow sheet for piping a
fermenter with an air filter. Many kinds of fibrous material can be
packed into the filtration vessels. Amor,g the more common are
cotton wool, slag wool, glass wool, and asbestos/cellulose fibers.
The bed depth should be at least 2.5 to 7.5 cm (usually more, and up
to 2 to 3 m deep for some commercial fermentations), and it should
be designed to produce a superficial velocity in the bed of 15 cm/s
(0.5 ft/s). The fiber diameter should be below 6 microns. This
will produce filtration efficiencies in the range of 99.7 to 100.0%
after 2 to 5 hours of operation. Filters can be regenerated every 2
to 5 hours by sterilizing with Itdry'' heat or steam at 160 to 180°C
f o r 2 hours, but they usually are regenerated only at the start of
each batch along with the fermenter.
Granular material, especially activated carbon and alumina, can
be used to sterilize air. Particle sizes are typically in the 30-
to 50-mesh range with filtration bed pressure drop up to 10 psi. The
efficiency will depend on the bed depth, air velocity, and degree of
contamination of the air. In some plants, after every 2 to 5 hours
o f operation, the filtration bed is taken off line and sterilized with
dry heat. In other plants, the bed is sterilized only before the
fermentation begins. The filtration mechanism for either granular
ORNL D W G 88-246
INLET AIR
STEAM
i FiLTER
FERMENTER Fig- J-1. P i p i n g diagram for fermenter
-- Source . G . B . Cherry and S . i). K f i x p , "The S t e Prozress in Industrial MicrobjDloLy, 4 37 ( 1 9 6
EXHAUST
air filtration. rilization of Ai 3).
r , "
155
or fibrous bed is a combination of several
simultaneously: (1) diffusion effects due to
electrostatic attraction between particles and
direct interception of a particle by the bed
mechanisms operating
Brownian motion, (2)
the bed material, ( 3 )
material, ( 4 ) inter-
ception as a result of inertial forces acting on the particle and
causing it to collide with the bed material, and (5) settling or
gravitational effects.
Sterilization of air by heating is technically feasible and is
probably the most certain method; however, it is often not applied for
economic reasons. At temperatures as low as 170 to 200"C, there is
an approximate exponential relation by which the time taken to kill
microorganisms decreases as the temperature to which they are exposed
increases. At 300°C, a 3-second exposure is sufficient.
The medium and equipment are normally sterilized with heat by
using steam. Sterilization with steam is a time vs temperature
optimization, as shown in Figure 5-2 which presents several time-
survival curves for steam sterilization. Usually 121°C for 15
minutes is sufficient, giving a probability of finding one viable
spore in 1000 sterilizations if each started with l o 5 spores. The
alternative cycle of 115°C for 30 minutes is only marginally adequate
due to an F, Factor of 7.5 (unit of lethality in the food industry
equivalent in minutes at 121°C of ail heat considered with respect
to its capacity to destroy spores o r vegatative cells of a particular
organism) giving the probability that 50% of the 1000 batches above
would have a viable spore. The fermentation brcth medium is normally
sterilized in the fermenter before introduction of the inoculum.
Figure J - 3 gives the two most common methods for controlling a
sterilization unit. Sterilization of the main fermenter is
accomplished with 145°C steam with a total turn around time of 10
hours.
Continuous sterilization of culture medium is practiced for
adding nutrients to ongoing fermentations and for heat sensitive
nutrients. Continuous sterilization can be done by injection of
steam or by using heat exchangers. Sterilization with steam injection
(Figure 5 - 3 ) is done by injecting steam into the nutrient solution
ORNL D W G 88-294
S O '
1 0 0
2
+ 0 U c LL
c3 z I
0 1 0 - 1
1 0 - 2
>
v)
5 10-3
1 0 - 4
10-5
----rT-- R A DiA T ION
0 10 20 30 40 50 0 0.2 0.4 0.6 0.8 1.0
HEATING TIME RADIATION DOSE A T 115OC (min) (Mrad)
A = LINEAR CURVE - CHARACTERISTIC OF MOST ORGANISMS
B = CURVE WITH SHOULDER FOLLOWED BY LINEAR REGION - CHARACTERISTIC OF MANY BACTERIAL SPORES
C = STEEP INITIAL CURVE FOLLOWED BY SHALLOWER CURVE A T LONGER TIMES
ACTIVATION = OCCURS WHEN SPORES ARE GERMINATED AT SHORTER TIMES (MOIST HEAT ONLY)
Fig. 5-2. Survival curves for sterilization of spores. S o u r c e : W . B . Hugo and A . D . Russell, P h a r m a c e u t i c a l M i c r o b i o l o g v , -.
B l a c k w e l l S c i e n t i f i c , London ( 1 9 7 7 ) .
157
ORNL D W G 88-250
STEAM
TEMPERATURE TRANSMITTER/ CONTROLLER
CONTROL VALVE
STEAM NOZZLE
MEDIUM - - HOT MEDIUM
CLOSED LOOP CONTINUOUS STERILIZER CONTROL SYSTEM
MEDIUM -
FLOW CONTROLLER 7
1 I I I I
-----
TEMPERATURE TRANSMITTER/ CONTROLLER TRANSMITTER
STEAM NOZZLE -
HOT MEDIUM
FEEDFORWARD-FEEDBACK, CONTINUOUS STERILIZER CONTROL SYSTEM
Fig. 5-3 . Continuous steam sterilization of medium feed. Source: G. R. Wilkinson and L. C . Baker, "Modern Trends in Steam Sterilization," Progress in Industrial Microbiolonv 5 231 ( 1 9 6 4 ) .
158
where the temperature is quickly raised to 140°C and maintained for
4 to 120 seconds. The added moisture is then removed by vacuum;
however, the process is sensitive to changes in viscosity of the
medium and to pressure variations. The procedure for using heat
exchangers is shown in Figure 5 - 4 . Over 90% of the energy input is
recovered, and the whole process takes only 3 to 4 minutes. Some
deposits do form on the heat transfer surfaces and are removed by
cleaning agents and the heat exchangers are resterilized.
159
ORNL DWG 88-251
CLEANING
CYCLE c *STERILE MEDIUM
TO FERMENTER
t - I
MEDIUM RESERVOIR
n --i
HEA TER x STEAM
Fig. 5-4. Continuous sterilization using heat exchangers. Source: Sterilization," ProEress in Industrial Microbiology 5 231 (1964).
G. R. Wilkinson and L. C. Baker, "Modern Trends in Steam
APPENDIX K
Equipment
163
Appendix K. EQUIPMENT
Antibiotic production equipment
This section addresses the specialized equipment required for
an antibiotics plant. Specifically included are the main fermenter
and auxiliary piping, aseptic metering pumps, agitator seals, and
specialized instrumentation.
A conventional fermenter (Fig. K - 1 ) usually consists of an
upright cylindrical tank fitted with four baffles, a jacket or coil
for heating and cooling, an air sparger, a device for mechanical
agitation, and an air filter (see appendix J). Basic instrumentation
consists of temperature, pH, and dissolved oxygen measurement and
control devices. While fermenters are constructed from many different
materials, 316 stainless steel clad vessels are almost always used
in the antibiotics industry. Figure K - 2 illustrates a typical
piping arrangement for sampling and inoculation of a fermenter
employed in the production of antibiotics.
Oxygen transfer is very important in antibiotic fermentations,
and this is normally accomplished with mechanical agitation. Figure
K - 3 illustrates several geometric configurations for a typical fer-
menter. It is essential, for aseptic operation, to seal the agitator
shaft as it enters the fermenter. This is usually accomplished with
a mechanical seal (Figs. K-4 and K - 5 ) . The stuffing box is usually
packed with graphite-asbestos and lubricated with silicone, mineral
oil, or antifoam oil. Since it is difficult to sterilize the packing,
there is an advantage to locating the stuffing box at least partially
within the fermenter to gain better heat transfer.
Aseptic metering pumps present some problems for long-term opera-
tion. Peristaltic pumps will provide aseptic operating conditions;
however, the flow is not continuous and the metering accuracy is not
great. Either valved pumps with the valves operated by cams and
springs, electromagnetic (mechanically linked to open and close)
gear, or rotary sleeve pumps are recommended.
Three areas of instrumentation which are crucial to the operation
of an antibiotic plant are dissolved oxygen concentration, pH, and
164
ORNL DWG 88-247
ANTlF RIENTS
COOLING COIL OR JACKET
D/T= 0.30-0.50 (.33) W/T= 0.08-0.12
(UNGASSED) Z / T = l .O-2.0 B/D =1 .O-2.5
L/D=0.25 H/D=0.5-1.0
Wi/D= 0.2 Fig. K-1. Standard fermenter diagra.
165
M I.” INOCULUM LINE I -
I I
ORNL DWG 88-249
4 STERILE AIR
STEAM STERILE AIR
FERMENTER
PRESSURE GAUGE +CU-D VENT
I 1 6 STERILE SA h, P L I N i
I I SAMPLE
SEED VESSEL
i FERMENTER I
/ + STEAM
SAMPLE PORT/DRAIN
SAMPLE/DRAIN 0 HARVEST LINE - STERILE FERMENTER INOCULATION FROM SEED TANK
Fig. K-2. Typical piping arrangement for sampling and inoculation.
166
ORNL DWG 88-240
f I
T
Q.5D
Z I T = 2
=1
Z IS UNGASSED
f
1 3D
I D
f D I T = 0.33
f I t
2.5D
0.5D
It”” 2.5D
t
Fig. K-3. Geometric configuration for agitator placement in a typical fermenter.
1 6 7
ORNL D W G 88-297
AGITATOR SHAFT /
F i g . K - 4 . Agitator shaft seal for a fermenter. S . A i b a , A . E. Humphrey, and N. F. Millis, Biochemical Source:
EnyineerinR, Academic Press, Inc., New York (1973).
168
ORNL D W G 88-295
BEARING HOUSING
CONDENSATE
3 FERMENTER HEAD
Y i ' " ' ' ' ' ' ' ' ' ' . ' . . . -
J A G I T SHAFT ATOR
Fig. K-5. Mechanical shaft seal for fermenter agitator. Source: S . Aiba, A . E . Humphrey, and N . F . M i l l i s , Biochemical EnJzineering, Academic Press , I n c . , New York ( 1 9 7 3 ) .
temperature. There are many excellent oxygen probes on the market.
Table K - 1 gives a list of the manufactures. In antibiotic processes,
it is desired to control the dissolved oxygen concentration at a set
level. Control of the dissolved oxygen concentration at a given
level can be achieved by altering the agitation rate, aeration rate,
oxygen partial pressure in the influent gas or the fermenter head
pressure, as indicated by the following equation:
where :
N, = Rate of 0, transfer by the fermenter.
K, = Overall 0, transfer coefficient.
a = Total interfacial area of gas bubbles in the fermenter.
P, = Partial pressure of 0, in the influent gas.
P, = Dissolved 0, concentration in the medium.
Temperature measurement and control are important and can be
accomplished with conventional equipment plus the addition of chilling
equipment for those locations which do not have cold (lO°C) ground
water for cooling. The importance of pH control has been well
established in antibiotic fermentations. The microorganisms can
secrete metabolites which will make drastic changes in the pH of the
medium leading to nonproductive conditions. The control of pH is
achieved by the automatic addition of acids (H,SO,) and/or bases
(NaOH) with metering pumps. The pH-sensing electrodes that are
inserted directly into a recirculating loop outside of the fermenter
are used to measure the pH of the medium.
I. 7 0
Table K-L. Suppliers of oxygen probes used in fermenters
Steam Oxygen C omD any Model No. -.-- Sterilizsble Ra-n=-
Ccir-Psrmer J - 5663-28
5-5663-00
Tngold Electrodes, Inc
Be ckman
Lee Scientific
Honeywell
Technology Inc
Electronic Ins t r Ltd.
Union Carbide
E. H. Sargent & Go.
Delta Scientific
777
100
55145-01,-02
P016OL. B
A1 5A
1101
S-38640-10B
75
Galvanic Yes 0 - 100%
Polarographic Yes 0- 100%
Polarographic Yes 0 - 100%
Polarographic Yes
Polarographic Yes
Polarographic No
Polarographic No
Galvanic No
Polarographic No
Galvanic No
Galvanic ?
0 - 100%
0 - 11 6mm. Hg PO,
0-14%
0-l5ppm
0-50 mg. 0, /liter
0 - 100%
APPENDIX L
Separation and Purification
1 7 3
Appendix L. SEPARATION AND PURIFICATION
This section will discuss two relatively new and potentially
very powerful separation/purification methods for antibiotics, pre-
parative chromatography and supercritical fluid extraction (SCF).
If the size of the antibiotic manufacturing process is to be substan-
tially reduced, the reduction will have to come in the separation
and purification section of the plant which represents a large por-
tion of a typical plant. Chromatography and SCF extraction are
relatively new separation techniques being applied to the antibiotic
process. Most of this work is still in the developmental stage,
although some penicillins and cephalosporins are separated commer-
cially using chromatography.
High Performance Liquid ChromatoEraDhv -
Figure L - 1 illustrates the many different types of chromatography
available. Figure L-2 presents a flow sheet for a commercial process
for separating p-lactam antibiotics (i.e., penicillins and cephalo-
sporins) using high performance liquid chromatography (HPLC). A
sample chromatogram is given in Fig. L-3 for the purification of
cephalosporin C (the most difficult separation for many antibiotics
is the removal of derivatives and other antibiotics). Tables L-1
and L - 2 summarize much of the work done on separation and purification
of antibiotics using HPLC. These tables give the necessary infor-
mation on the operation of the HPLC for many different antibiotics.
The disadvantages o f HPLC are that it is a batch process, it
causes dilution of the product, and it is difficult to scale up due
to the flow dynamics in the narrow bore columns which are required.
These problems were solved with the introduction of the annular
chromatograph depicted in Fig. L-4.51 This device uses the same
exact resins which are used in HPLC columns except that now the
material is packed into the narrow rotating annulus. The rotation
is timed to be in phase with the feed rate so that each component
inthe feed exits the adsorption bed at a specific point, thus making
the process continuous and of much greater utility from a processing
QRNL DWG 88-255
CHROMATOGRAPHY
$.\
GAS -LIQUID A GAS-SOLID COLUMN A OPEN BED
LIQUID-SOLID BONDED-PHASE EXCLUSION
A GEL- PE R M E ATlO N GEL- FILT R A T IO N
Fig. L - 1 . Schematic diagram showing the many types of chromatography that are in use.
175
ORNL DWO 81-474
WASH SOLVENT
CRUDE CEFONlClD
DIAFLTRATON
SATURATOR
MOBLE PHASE
ONEXCHANGE CARBONFLTER ULTRAFLTER
J DETECTOR
WASTE COLLECTED FRACTION
Fig. L-2. Process chromatograph flow diagram. Source: A. l?. Cantwell, R . Calderone, aid ?f. Sienko, "Process Scale-up of a /?-Lactam Antibiotic Purification by High-Performance Liquid Chromatography," Journal of Chromatographv. 316 133 (1984).
176
ORNL D W G 88-298
U.V. RANGE: 254 nm, 0.08 AUFS CHART SPEED: 5 mm/min FLOW RATE: 1.5 ml/min COLUMN SIZE: 2x250 mm PACKING: p NH2 (VARIAN AEROGRAPH) S 0 LV E N T: HO Ac-C H 3 0 H-CH 3C N-H 2 0 (2 1417.5186.5) P.S.I.: 3400
2
INJECT 16 min
1. DEACETOXYCEPHALOSPORIN C, 1.5 Fg 2. DEACETYLCEPHALOSPORIN C, 1.5 pg 3. 5-METHYCEPHALOSPORIN C, 1.5 p g 4. CEPHALOSPORIN C, 2.0 p g
Fig. L-3. Sample chromatogram for the separation of cephalosporin C .
177
Table L-1. Chromatographic parameters for tetracyclines
Chromatographic Support Mobile phase Comments system and column
Ion Exchange
Adsorption Chromatography
Liquid- liquid Partition Chromatography
Reversed- Phase Partition Chromatography
Zipax SCX
Corasil Coated with Pol ye thylene glycol 400
Oc tadec yl silane Bonded on
0.4u K,B,O, ,O.Olu EDTA 1% isopropyl alcohol efficiency
Poor column
(PH 9.8)
0.m citric acid, Very poor 0. O O m EDTA pH 5.2 e f f ic iency
Aqueous 0.08Ij HC10, Broad over- lapping peaks
0. l I j HC10, , 1.9u Good NaC10, , 0.005Ij separation citric acid- acetonitrile ( 2 : l )
Dioxane-pentane (1.5:l) Poor column efficiency Poor repro- duc ib il i ty
0 . u HC10, , 0.4u NaC10, 0.0025N citric acid- efficiency acetonitrile (85:15 v/v)
Poor partition
Partisil (18~)
178
Table L-2. HPLC assays of antibiotics
Recovery "Stationary Mode ot Sensit: * ' i - Llrli, Cxt r ac t I on ( X ) Mobil phase Phase Separation Detection (mIiJt
Partition/ W absorption 0.3
Reverse phase Methanol- c-18
Sodium acetate Cef amandole methanol- ---
85+
95-100
= l o o %
98
_ _ _
97f
92-100
sodium acetate
Trichloroacetic Methanol- Pnenylsilane Partition/ W absorption 1.0
Acetic acid Reverse phase Cefazolin
Cefuroxime
Cephalexin
Cephaloridine
Cephalothin
Cephalothin
Cephr adine
Acetic acid- C - 1 8 Partition/ W absorption 1.0
methanol-water Reverse phase Dimethylformamide
Methanol Methanol-water C-18 Partition/ UV absorption 0.5
Reverse phase
_ . \ . I, Trichloroacetic
acid
Methanol- Phenylsilane Partition/ W absorption 2.0
ammonium acetate Reverse phase
Trichloroacetic
acid
Methanol- Phenylsilane Partition1 W absorption 10.0 ammonium acetate Reverse phase
Methanol- c-18 acetic acid
Partition/ W absorption 1.0
Reverse phase Dimethylformamide
Ammonium c-18
carbonate
Partition/ W absorption 0.5 Reverse phase
None
Ampicillin/ Perchloric acid --- Arnoxicillin
Methanol- C-8
Phosphate buffer
Partition/ W absorption 0.5
Reverse phase
_-_ Penicillins 0 . 0 5 t j phosphate C-18
Acetonitrile
Partition/ UV absorption Reverse phase
Tetracyclines Partition/ UV absorption Reverse phase
55% acetonitrile1 C-18
45% 0 . 0 5 t j phosphoric acid
Erythro;nycin Diethyl ether 96+ Acetronitrile- C-18
Ammonium acetate-
water
Partition/ U Fluorometry 0.1
Reverse phase
is Octadecyl silane bonded phase, C-8 is Octyl silane bonded phase
1 7 9
ORNL DWG 74-1263814
GAS OVERPRESSURE 1
CONTINUO
STATIONARY ELUENT INLET INLET DISTRIBUTOR
INLET PRESSURE SEAL
FEED INLET
FEED STREAM
ROTATING ANNULAR CHROMATOGRAPH \
STATIONARY ELUENT WASTE COLLECTION
ELUATE EXIT i
ELUENT STREAMS
SEPARATED CONSTITUENTS
PRODUCT COLLECTION
ANNULAR BED
Fig. L - 4 . Schematic diagram of continuous rotating annular chromatograph.
standpoint. The use of an annulus for the resin instead of a packed
bed eliminates the scale-up problems with Sed compression as the
column diameter is increased.35 Recently the Oak Ridge National
Laboratory (ORNL) has demonstrated that annular chromatography can
concentrate products in addition to separating them. There is a
wealth of experimental data in the literature on the selectivity of
resins for antibiotic separation which has been evaluated in HPLC
columns. These same data can be used with the aid of mathematical
models developed at ORNL for evaluating the performance of other
annular chromatographs with these same resins.52 Annular chromatog-
raphy has the potential to effect a vast decrease in the size and
complexity of the separation and purification of antibiotics on a
scale comparable with the great advances made in the 1940s by the
submerged culture process for the fermentation of antibiotics.
Several large-scale pilot units of the annular chromatograph are
available at ORNL. Because these units have a very high capacity,
it would be possible during a national emergency to skid-mount an
annular chromatography system on a truck and simply drive it to any
emergency production facility.
Supercritical Fluid Extraction
The solubility of solids in SCF was first discovered over 100
years ago. Recently SCF extraction w a s applied commercially to the
decaffination of coffee and has received widespread interest in the
pharmaceutical industry because this separation technique can be
performed at low temperatures (20 to 60°C). The advantages are
obvious. SCF extraction operates like distillation or liquid/liquid
extraction but at lower temperatures and with milder less toxic
solvents. Figure L-5 presents a crude flow sheet for the process.
The separation and purification of antibiotics with SCF extraction
has been patented in Germany.41 Carbon dioxide is the solvent of
choice because of its availability, nontoxicity, and proper thermo-
dynamic properties. The solubility of most antibiotics in SCF CO,
is low; however, if 5 to 10% of a polar second solvent is added,
these solubilities can increase by one or two orders of magnitude.53
181
ORNL DWG 88-254
PURIFIED ANTIBIOTIC 4
EXPANSION
WASTE BROTH 'POLAR ORGANIC
MAKE-UP HIGH PRESSURE
HIGH PRESSURE PUMP
Fig. L-5. Schematic diagram for supercritical f luid extraction using CO, .
182
A unique property of this process is that the dissolved substance
may be separated by decreasing the fluid density by either decreasing
the pressure or increasing the temperature. The key point is that a
small change in temperature or pressui2 will result in a large
chan5e in solvent density and thus a large change in solubility.
The process can be easily staged so as to provide enhanced recovery
with a small expenditure of energy.35g42 The pressures required are
in the same range as the high pressilre steam which is found at most
industrial manufacturing facilities.
Conventional liquid-liquid-extraction, which is quite common in
the antibiotic industry, often requires a difficult crystallization,
a distillation and/or use of a second solvent. A solvent residue
may be left on the product thus requiring further purification.
With SCF extraction, the extract is separated by reducing the solvent
density to the gaseous state, thereby leaving very little solvent on
the product. In addition, the transport properties of S C F ' s are
closer to those of the gaseous state than the liquid state, giving
rise to enhanced diffusivities and viscosities which will minimize
any mass transfer problems in the extraction. Recently, a German
patent has been issued for the separation and purification of anti-
biotics using supercritical fluids. The major advantages would be a
reduction in the number of processing steps and the elimination of
the large and expensive solvent recycle system.
Many different SCF solvents are possible as extraction agents.
The best solvents can often be easily identified from thermodynamic
data which is rapidly becoming available in the open literature.
For example, from Table L - 3 , the solubility of penicillin V in
ethanol is over twice what it is in water (the major constituent of
the fermentation broth). In the supercritical region ethylene and
water are immiscible, with ethanol distributing between the two
phases. It is reasonable to assume that if a SCF mixture of ethylene
and ethanol were used for fermentation broth extraction, penicillin
V would be concentrated in the SCF phase. An exact prediction of the
final result can be made if the thermodynamic data are used to tune
a mathematical model known as an equation of state.
183
Table L - 3 . SCF related physical properties of selected antibiotics
Antibiotic Molecular Melting Solubility in Weight Point ("C) Water Ethyl E thano 1
Acetate
PENICILLIN V 3 5 0 . 0 124 .0 9 . 0 >20.0 20 .0 (Phenoxymethyl penicillinic acid)
K salt 388 .O 2 6 3 . 0 >20.0 0 . 6 5 1 .34
PENICILLIN G 3 3 4 . 0 (Benzyl -
Na salt 356.0 215.0 >20.0
K salt 372 .O 2 1 5 . 0 >20,0
penicillin)
TETRACYCLINE 4 4 4 . 0 1 7 2 . 5 1 . 7
OXYTETRACYCLINE 4 6 0 . 0 182 .0 0.60
0 . 4 0 9 . 9 7
0 . 4 2 10.4
1 7 . 3 >20.0
0 . 8 5 8 . 1
CEPHALOSPORIN C 4 1 5 . 4
CEPHALOTHIN 3 8 2 . 4 1 6 0 . 5
Na salt 4 0 4 . 4 >20.0 0.02 17.7
CEPHALORIDINE 399.0 >20.0 0.035 1 . 9 5
BACITRACIN 1 4 2 1 . 0 >20.0 0 . 0 4 7 9 . 1
aSolubility in g/1 at 2 9 4 - 3 0 1 "C
185
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Almannavarnir Rikisins Reykj avic IC ELAND
Stato Maggiore Difesa Civile Centro Studi Difesa Civile Rome ITALY
Directeur de la Protection Civile Ministere de 1’Interieur 36 Rue J. B. Esch Grande-Duche de LUXEMBOURG
Directeur Organisatrie Bescherming Bevoling Ministry of Interior Schedeldoekshaven 200 Postbus 20011 2500 The Hague THE NETHERLANDS
The Head of Sivilforsvaret Sandakerveien 12 Postboks 8136 Oslo dep Oslo 1 NORWAY
Servicio National de Proteccao Civil Rua Bela Vista a Lapa, 57 1200 Lisbon PORTUGAL
Jefe, Seccion de Estudios y Planificacion c/Evaristo San Miguel, 8 Madrid- 8 ESPANA
Civil Defense Administation Ministry of Interior Ankara TURKEY
Home Office Scientific Advisory Branch Horseferry House Dean Ryle Street London SWlP 2AW ENGLAND
188
104
105
Mr. Eric E. Alley, Esquire President The Institute of Civil Defence Bell Court House Blomfield Street London EC2M 7AY ENGLAND
Civil Emergency Planning Directorate North Atlantic Treaty Organization 1110 NATO BELGIUM
Additional Di s tr i.but ion
106.
107.
108.
109.
110.
111.
112.
The American Civil Defense Association (TACDA) P.O. Box 1057 Starke. FL 32091
Dr. William Anderson Division of Fundamental Research for
National Science Foundation 1800 G Street, N.W. Washlngton, D.C. 20550
Emerging and Critical Engineering Systems
Ivars Gutmanis, President Sterling-Hobe Corp. 910 Seventeenth Street, N.W. - Suite 309 Washington, DC 20006
Nolan Jones, Ph.D. National Governors Association 444 North Capitol Street Washington, DC 20001
Richard K. Laurino, Pres. Center for Planning and Research 2483 East Bayshore Road Palo Alto, CA 94303
Prof. Jiri Nehnevaj sa Department of Sociology 2L22 Forbes Quadrangle University of Pittsburgh Pittsburgh, PA 15260
Joseph Russo 8613 Melwood Road Bethesda, MD 20817
189
113.
114.
115.
M s . Joanne Shore I EAL 2600 Virginia Ave., N.W. Washington, DC 20037
Walmer E. Strope Center for Planning and Research 5600 Columbia Pike, Suite 101 Bailey's Crossroads, VA 22041
Chuck Wilton Scientific Services, Inc 517 East Bayshore Redwood City, CA 94603
116
117
118
119
120
Federal Emergency - Coordinators
Craig Alderman, Jr. Director, Emergency Planning Office of the Secretary of Defense Department of Defense Washington, DC 20310-2200
William Gillen Emergency Preparedness Manager DHHS 3B 10 Hubert Humphrey Bldg. 200 Independence Avenue, S.W. Washington, DC 20201
Kent Gray CDC ERCG 1600 Clifton Road, N.E. - M S - F 3 8 Atlanta, GA 30333
Bruce Pelton Emergency Coordinator U.S. Department of Commerce Office of the Secretary Room 5044 HCHB Washington, DC 20230
Thomas P. Reutershan Emergency Coordinator U.S. Public Health Service 5600 Fishers Lane - Rm 4-81 Rockville, MD 20857
190
121.
122.
123,
124
125
126
OVERSEAS
Prof. Dr. Lars Clausen Soziologisches Seminar d e r
Olshausenstrasse 40/60 D-2300 Kiel FEDERAL REPUBLIC OF GERMAXY
Christian-Albrechts-Universitat
Mrs. F. Lorraine Davies Director, Emergency Services Medical Services Branch Health and Welfare Canada 11th Floor Jeanne Mance Building Tunrrey ' s Pasture Ottawa, Ontario K1A OL3 CANADA
Ross Lunn Office of International Affairs Emergency Planning Canada Gillin Building 141 Laurier Avenue, West Ottawa KlAOW6 Ontario, CANADA
Carlo Pelanda Istituto di Sociologia Internazionale I34170 Gorizia - Via Malta, 2 Italia Tel. (0481) 83632
Dr. Thomas Ritchey National Defence Research Institute Defence Analysis Department P.O. Box 27322 S-102 54 STOCKHOLM, Sweden
Lloyd Swick Principal Consultant for Pub 1 ic Pro t e c t ion
Emergency Planning Canada Gillin Building 141 Laurier Avenue, West Ottawa KlAOW6 Ontario, CANADA
127-284. Given distribution as shown in Category UC-41.
Unclassifted April 1988 April 1988
200 pages
EXPEDIENT ANTIRIOTICS PRODUCTION -- 1988 Unclassified EXPEDIENT ANTIRIOTICS PRODUCTION -- 1988
200 pages by P. R. Bienkowski, C. 11. Eyers, and D. D. Lee by P. R. Bienkowski, C. H. Byers, and D. D. Lee Oak Ridge National Laboratory, Oak Ridge, TN 37831 Oak Ridge National Laboratory, Oak Ridge, TN 37831 Interagency Agreement: FEMA No. EMW-84-E-1737; DOE No. 1457-1457-AI Interagency Agreement: FEMA No. EMW-84-E-1737; DOE No. 1457-1457-A1
The literature o n the Fanufacture, separation and purification, and clinical uses of antibiotics was revtewed, and a bibliography of the pertinent material was compiled. Five antimicrobial drugs, penicillin V and G , cephalosporin, tetracycline, Bacitracin, and sulfonamide, were identified for emergency production. Antibiotics plants I n the United States, Mexico, and Puerto Rico and potential alternate sites such as single cell protein, enzyme, and fermentation ethanol plants were identified.
specialized sterile equipment and an antibiotic-specific separation and purification unit. reduction i n the complexity and degree of separation and purity normally required. Detailed instructions and recommendations were developed to assist State and Federal officials in directing the resumption of antibiotic production. Alternate sites require a team of skilled personnel to convert to antibiotic production Ln the austere environment.
A n t i b i o t i c - m a n u f a c t u r i n g plants contain a generic fermentation unit with
Feasible emergency production will require a substantial
The literature on the manufacture, separation and purification, and clinical uses of antibiotics was reviewed, and a bibliography of the pertinent material was compiled. Five antimicrobial drugs, penicillin V and G , cephalosporin, tetracycline, Bacitracin, and sulfonamide, were LdentiEied f o r emergency production. and potential alternate sites such as single cell protein, enzyme, and fermentation ethanol plants were identiEied.
specialized sterile equipnent and an antibiotic-specific separation and purification unit. reduction in the complexity and degree of separation and purity normally required. State and Federal officials in directing the resumption of antibiotic production. t o antibiotic production in the austere environment.
Antibiotics plants i n the United States, Mexico, and Puerto Rico
A n t i b i o t i c - m a n u f a c t u r i n g plants contain a generic fermentation unit with
Feasible emergency production will require a substantial
Detailed instructions and recommendations were developed to assist
Alternate sites require a team of skilled personnel to convert
EXPEDIENT ANTIRIOTICS PRODUCTION -- 1988 EXPEDIENT ANTIRIOTICS PRODUCTION -- 1988 April 1988
200 pages by P. R. Bienkowski, C. 11. Ryers, and D. D. Lee Oak Ridge National Laboratory, Oak Ridge, TN 37831 Interagency Agreement: FEMA No. EMW-84-E-1737; DOE No. 1457-1457-A1
The literature on the manufacture, separation and purification, and clinical uses of antibiotics was reviewed, a n d a bibliography of the pertinent material was compiled. Five antimicrobial drugs, penicillin V and G , cephalosporin, tetracycline, Bacitracin, and sulfonamide-, were identified for emergency production. Antibfottcs plants i n the United States, Mexico, and Puerto Rico and potential alternate sites such as single cell protein, enzyme, and fermentatton ethanol plants were identified.
specialized sterile equipment and an antibiotic-specific separation and purification unit. Feasible emergency production will require a substantial reduction in the complexity and degree of separation and purity normally required. Detailed instructions and recommendations were developed to assist State and Federal officials in directing the resumption of antibiotic production. Alternate sites require a team of skilled personnel to convert t o antibiotic production i n the austere environment.
A n t t b i o t i c - m a n u f a c t u r i n g plants contain a generic fermentation unit with
by P . R. Bienkowski, C. 11. Eyers, and D. D. Lee Oak Ridge National Laboratory, Oak Ridge, TN 37831 Interagency Agreement: FEMA No. EMU-84-E-1737; DOE No. 1457
Unclassified April 1988
200 pages
.14 5 7-A 1
The ltterature on the manufacture, separation and purification, and clinical uses of antibiotics was reviewed, and a bibliography of the pertinent material was compiled. Five antimicrobial d s , enicillin V and G , ce halosporin, tetracycline, Bacitracin, a m n a m i d e f were identified for :mergencx production. Antibiotics plants in the United States, Yexico, and Puerto Rico and potential alternate sites such as single cell protein, enzyme, and fermentatton ethanol plants were identified.
specialized sterile equipoent and an antibiotic-specific separation and purification unit. Feasible emergency production will require a substantial reduction in the complexity and degree of separation and purity normally required. Detailed instructions and recommendations were developed to assist State and Federal officials in directing the resumption of antibiotic production. Alternate sites require a team of skilled personnel to convert to antibiotic production in the austere environment.
A n t i b i o t i c - m a n u f a c t u r i n g plants contain a generic fermentation unit with
